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Abstract:

Provided are biosensors, compositions comprising biosensors, and methods
of using biosensors in living cells and organisms. The biosensors are
able to be selectively targeted to certain regions or structures within a
cell. The biosensors may provide a signal when the biosensor is targeted
and/or in response to a property of the cell or organism such as membrane
potential, ion concentration or enzyme activity.

Claims:

1-36. (canceled)

37. A ligand-dye complex, comprising a cognate ligand of a dye non
covalently bound to the dye, wherein the cognate ligand comprises an
antibody fragment comprising a polypeptide sequence having at least 90%
sequence identity to the polypeptide of one of SEQ ID NO: 13, wherein the
dye that binds the antibody fragment comprises one of a malachite green
or an analog of malachite green, and wherein the bound dye and ligand
exhibit an increase in detectable fluorescence signal at least ten times
greater than the detectable fluorescence signal of the dye when not bound
to the ligand.

38. The ligand-dye complex of claim 37, wherein the ligand-dye complex
further comprises the antibody fragment bound to a protein.

39. The ligand-dye complex of claim 37, wherein the bound dye and ligand
exhibit an extinction coefficient of greater than 30,000 and a maximum
absorption at a wave length of greater than 350 nm.

40. The ligand-dye complex of claim 37, wherein the bound dye emits
photons having a changed polarization upon binding the ligand.

41. The ligand-dye complex of claim 40 wherein the polarization change is
greater than or equal to 20%.

42. The ligand-dye complex of claim 37, wherein the dye of the bound dye
and ligand exhibits an increased emission wave length of at least 10 nm.

43. The ligand-dye complex of claim 37, wherein the ligand is fixed to a
substrate.

44. The ligand-dye complex of claim 43, wherein the substrate comprises
molecules of formula X(a)-R(b)-Y(c), wherein R is a spacer, X is a
functional group that binds R to a surface, Y is a functional group for
binding to the ligand, (a) is an integer from 0 to about 4, (b) is an
integer from 0 or 1, and (c) is an integer not equal to 0.

45. The ligand-dye complex of claim 37, wherein the ligand-dye complex
occupies a microenvironment and wherein the dye provides the increase in
detectable fluorescence signal in response to pH, polarity, restriction,
or mobility properties within the microenvironment.

46. A method of forming a ligand-dye complex for use in detecting
expression of a gene in a cell, the method comprising: adding a vector to
a cell, wherein the vector comprises a nucleic acid gene encoding an
antibody fragment comprising a polypeptide sequence having at least 90%
sequence identity to the polypeptide of SEQ ID NO: 13; causing expression
of the gene to produce a gene product comprising the antibody fragment;
adding dye to the cell to bind to the gene product, wherein the dye
comprises one of a malachite green or an analog thereof.

47. The method of claim 46, wherein the gene further encodes a second
protein, and wherein the gene product comprises a fusion protein
comprising the antibody fragment and the second protein.

48. The method of claim 47, wherein the dye is substantially excluded
from an interior of the cell, and wherein fluorescence is detected only
when a portion of the second protein that is fused to the antibody
fragment is exposed at a surface of the cell.

49. A method of forming and using a ligand-dye complex for detecting
interaction of two proteins in a cell comprising: adding a vector to a
cell, wherein the vector comprises a nucleic acid gene encoding a first
protein fused to a first portion of a polypeptide sequence having at
least 90% sequence identity to the polypeptide of SEQ ID NO: 13; adding a
vector to a cell, wherein the vector comprises a nucleic acid gene
encoding a second protein fused to a second portion of the polypeptide
sequence; causing expression of the genes to produce fusion proteins
comprised of the first and second proteins fused to first and second
portions, respectively, of the polypeptide; and adding dye to the cell,
wherein the dye comprises one of malachite green or an analog thereof;
and detecting a fluorescence signal indicative of the dye binding to the
fusion proteins, wherein the fluorescence signal is at least ten times
greater than the detectable fluorescence signal of the dye when not bound
to the fusion proteins.

50. The method of claim 49, wherein the first polypeptide portion and the
second polypeptide portion interact to form a molecule comprising a
polypeptide sequence having at least 90%-sequence identity to the
polypeptide of SEQ ID NO: 13, and wherein fluorescence is detectable when
interaction between the first protein and the second protein brings the
first polypeptide portion into proximity of the second polypeptide
portion.

Description:

PRIORITY

[0001] This application is a division of co-pending U.S. application Ser.
No. 12/524,328 which has a section 371(c) date of Feb. 3, 2010, and which
is a U.S. National Stage application based on International Application
Serial No. PCT/US2008/051962 filed 24 Jan. 2008 and claims the benefit of
U.S. Provisional Application Ser. No. 60/897,120 filed Jan. 24, 2007 and
U.S. Provisional Application Ser. No. 61/013,098 filed Dec. 12, 2007, the
contents of each of which are incorporated by reference in their
entirety.

REFERENCE TO SEQUENCE LISTING

[0003] This application contains a Sequence Listing in accordance with 37
C.F.R. §§1.821-1.825. The material in the Sequence Listing text
file is herein incorporated by reference in its entirety in accordance
with 37 C.F.R. §1.52(e)(5). The Sequence Listing, entitled
"070683PCTUS_Jul. 24, 2012--5 T25.txt", contains one 111 Kb text
file and was created on Jul. 22, 2009 and amended on Jul. 24, 2012 using
an IBM-PC machine format.

BACKGROUND

[0004] The identification, analysis and- monitoring of biological analytes
(such as polypeptides, polynucleotides, polysaccharides and the like) or
environmental analytes (such as pesticides, biowarfare agents, food
contaminants and the like) has become increasingly important for research
and industrial applications. Conventionally, analyte detection systems
are based on analyte-specific binding between an analyte and an
analyte-binding receptor. Such systems typically require complex
multicomponent detection systems (such as ELISA sandwich assays) or
electrochemical detection systems, or require that both the analyte and
the receptor are labeled with detection molecules (for example
fluorescence resonance energy transfer or FRET systems).

[0005] One method for detecting analyte-binding agent interactions
involves a solid phase format employing a reporter labeled
analyte-binding agent whose binding to or release from a solid surface is
dependent on the presence of analyte. In a typical solid-phase sandwich
type assay, for example, the analyte to be measured is an analyte with
two or more binding sites, allowing analyte binding both to a receptor
carried on a solid surface, and to a reporter-labeled second receptor.
The presence of analyte is detected based on the presence of the reporter
bound to the solid surface.

[0006] A variety of devices for detecting analyte/receptor interactions
are also known. The most basic of these are purely chemical/enzymatic
assays in which the presence or amount of analyte is detected by
measuring or quantitating a detectable reaction product, such as gold
immunoparticles. Analyte/receptor interactions can also be detected and
quantitated by radiolabel assays. Quantitative binding assays of this
type involve two separate components: a reaction substrate, e.g., a
solid-phase test strip and a separate reader or detector device, such as
a scintillation counter or spectrophotometer. The substrate is generally
unsuited to multiple assays, or to miniaturization, for handling multiple
analyte assays from a small amount of body fluid sample.

[0007] Biosensor devices integrate the assay substrate and detector
surface into a single device. One general type of biosensor employs an
electrode surface in combination with current or impedance measuring
elements for detecting a change in current or impedance in response to
the presence of a ligand-receptor binding event. This type of biosensor
is disclosed, for example, in U.S. Pat. No. 5,567,301. Gravimetric
biosensors employ a piezoelectric crystal to generate a surface acoustic
wave whose frequency, wavelength and/or resonance state are sensitive to
surface mass on the crystal surface. The shift in acoustic wave
properties is therefore indicative of a change in surface mass, e.g.; due
to a ligand-receptor binding event. U.S. Pat. Nos. 5,478,756 and
4,789,804 describe gravimetric biosensors of this type. Biosensors based
on surface plasmon resonance (SPR) effects have also been proposed, for
example, in U.S. Pat. Nos. 5,485,277 and 5,492,840. These devices exploit
the shift in SPR surface reflection angle that occurs with perturbations,
e.g., binding events, at the SPR interface. Finally, a variety of
biosensors that utilize changes in optical properties at a biosensor
surface are known, e.g., U.S. Pat. No. 5,268,305.

[0008] All of the above analyte detection systems are characterized by the
requirement for a secondary detection system to monitor interactions
between the analyte and the receptor. A need still exists for a direct,
homogeneous assay for analyte detection, i.e., one that may be used in
living cells, which will be more versatile in terms of the range of
applications and devices with which it can be used.

SUMMARY

[0009] Provided are biosensors, compositions comprising biosensors, and
methods of using biosensors in living cells and organisms. The biosensors
are able to be selectively targeted to certain regions or structures
within a cell. The biosensors may provide a signal when the biosensor is
targeted and/or in response to a property of the cell or organism such as
membrane potential, ion concentration or enzyme activity.

[0010] In one embodiment, the biosensors comprise at least two components;
(1) a selectivity component capable of interacting with a target molecule
of interest and a (2) reporter molecule that produces a detectable change
in signal upon interaction of the selectivity component with the target
molecule. The reporter molecule may be covalently linked to the
selectivity component, or it may be able to noncovalently interact with
the selectivity component. In certain embodiments, the selectivity
component is a ligand of a reporter molecule such as a dye.

[0011] The selectivity component, which in certain embodiments is
expressed within the cell or organism to be analyzed, may be a
polypeptide (including antibodies and non-antibody receptor molecules,
and fragments and variants thereof), polynucleotide (including aptamers),
template imprinted material, or organic and inorganic binding element.
The selectivity component may be biologically selected to favor reporter
molecule binding and sensitivity. The selectivity component may bind
directly to a target molecule, or be fused to a targeting moiety (such as
a protein) that binds to the target molecule.

[0012] The reporter molecule may be sensitive to changes in the
environment, including, for example, pH sensitive molecules, polarity
sensitive molecules, restriction sensitive molecules, or mobility
sensitive molecules. The reporter molecule may, in embodiments where in
it noncovalently interacts with the selectivity component, comprise an
additional moiety that binds to the selectivity component.

[0013] The biosensor may optionally comprise a chemical handle suitable to
facilitate isolation, immobilization, identification, or detection of the
biosensors and/or which increases the solubility of the biosensors.

[0014] In one example, the selectivity component is a single chain
antibody (scFv) that comprises amino acid sequences that lead to specific
binding of certain reporter molecules such as monomethin cyanine dyes
(TO1 and its analogs). In yet other embodiments, the selectivity
component is a single chain antibody that comprises amino acid sequences
that lead to specific binding of a reporter molecule such as Malachite
Green (and its analogs). The formation of such protein-dye complexes
produces a large increase in fluorescence of the dye (i.e., fluorogen
reporter molecule) when it is in the bound state, thereby allowing
detection of binding. In other examples, the single chain antibody is
coupled (e.g., as a fusion protein or chemical conjugate) to a molecule
of interest, such as for example a cell cycle regulatory protein.

[0015] In certain other embodiments, a binary biosensor is used to detect
a molecule of interest. For example, a VH chain of a dye-specific
antibody can be conjugated to a lipid, sugar, protein or polypeptide of
interest, while the corresponding VL chain can be coupled to another
potential ligand or polypeptide of interest. When the target and ligand
are in close proximity, the VH and VL chains become close
enough to form a binding epitope for the dye, a detectable signal is
produced.

[0016] The biosensors described herein are useful for both in vivo and in
vitro applications. In various embodiments, the biosensors may be used
for detecting one or more target molecules, detecting environmental
pollutants, detecting chemical or biological warfare agents, detecting
food contaminants, and detecting hazardous substances. In an exemplary
embodiment, the biosensors may be used for intracellular monitoring of
one or more target molecules. In such embodiments, at least one component
of the biosensor may be expressed within the cell to be analyzed.

[0022] FIG. 6 depicts the structure of Malachite Green derivatized with a
PEG amine. In some embodiments, the amino group may be covalently
modified with a biotin group for streptavidin coated magnetic bead
enrichment of yeast bearing scFv proteins that bind to the Malachite
Green on the opposite end of the PEG linker.

[0023] FIG. 7 depicts the structure of Thiazole Orange 1 (TO1) derivatized
with a PEG amine. In some embodiments, the amino group may be covalently
modified with a biotin group for streptavidin coated magnetic bead
enrichment of yeast bearing scFv proteins that bind to the TO1 on the
opposite end of the PEG linker.

[0027] FIGS. 11A, 11B and 11C illustrate fluorogen embodiments and their
use with yeast displayed scFvs. FIG. 11A depicts the structure of the
fluorogenic dyes thiazole orange derivative (TO1-2p) and malachite green
derivative (MG-2p) used in various embodiments. FIG. 11B is a graph
depicting the isolation of FBPs using FACS. The Sorting screen shows
separation of yeast cells bearing malachite green-activating scFvs from a
bulk yeast population. The horizontal axis shows distribution of cells by
green fluorescence of antibody reagent that labels the c-myc epitope; and
the vertical axis depicts distribution of cells by red fluorescence
generated by binding of MG fluorogen. Sorting window (I) collects cells
enriched for FBPs composed of heavy chain (VH), light chain (VL) and
c-myc epitope (M). Sorting window (II) collects cells enriched for FBPs
composed only of the heavy chain. FIG. 11C is a graph depicting a
homogenous format assay of live yeast cells displaying FBPs. The
fluorescence excitation spectrum of displayed HL4-MG is taken on a
96-well microplate reader (107 yeast cells in 200 ml effective
concentration B10 nM scFv are treated with 200 nM MG-2p). The inset
illustrates low levels of fluorescence background signal with JAR200
control cells that do not express FBPs.

[0028] FIG. 12 depicts the improvement of binding affinity and intrinsic
brightness of HL1-TO1 by directed evolution. Affinity and total cellular
brightness are measured using yeast cell surface displayed scFvs. Total
cellular brightness is measured at saturating fluorogen concentration on
a Tecan Safire 2 plate reader, and intrinsic brightness calculated by
normalizing total signal to the relative number of scFvs, determined
separately by FACS analysis of immunolabeled c-myc epitope. The bar graph
depicts relative intrinsic brightness for selected scFvs employed after
one or two generations of directed evolution. Numbers on the bars
represent cell surface binding KD (nM). The sequence alignments show
the distribution of acquired mutations within the heavy chain variable
region of HL1-TO1 (SEQ ID NO: 73, a fragment corresponding to residues
1-121 of SEQ ID NO: 3). Complementarity Determining Regions ("CDRs")
within HL1-TO1 implicated in antigen recognition are underlined and
numbered 1, 2 and 3, (corresponding to SEQ ID NOs 76, 77 and 78,
respectively) as identified in the IMGT/V-QUEST database. Amino acid
replacements in bold depict residues found in multiple instances within
each family of improved descendants (a fragment of HL1.1-TO1, SEQ ID NO:
74 and a fragment of HL1.0.1-TO1, SEQ ID NO: 75). The dominant
replacements tend to accumulate in CDRs, wherein replacement CDRs within
the fragment of HL1.1-TO1 shown to align with SEQ. ID NOs: 77 and 78 are
represented by SEQ ID NOs: 79 and 80 and replacement CDRs within the
fragment of HL1.0.1-TO1 shown to align with SEQ. ID NOs: 77 and 78 are
represented by SEQ ID NOs: 81 and 82. For HL1.1-TO1, accumulation of
dominant replacements occurs in the heavy chain rather than the light
chain. Among 16 unique second generation descendants that are analyzed, 8
positions in the heavy chain accumulate dominant mutations but only 1
position in the light chain accumulates dominant mutations. For the
selected clones, it can be seen that the first generation replacements
improve both affinity and brightness, whereas second generation
replacements improve only affinity.

[0029] FIG. 13 is an SDS-PAGE gel of purified FBPs with 1 μg of BCA
quantitated scFv loaded per lane, where lane-1 is loaded with a MW
standard; lane-2 is loaded with HL1.0.1-TO1; lane-3 is loaded with
HL4-MG; lane-4 is loaded with L %-MG; and lane-5 is loaded with H6-MG.

[0031] FIG. 15 provides graphs illustrating absorbance of fluorogens and
FBP/fluorogen complexes. Shown are samples used in quantum yield
determinations. The absorbance of FBP/fluorogen complexes is obtained on
a dual beam PerkinElmer Lambda 45 spectrophotometer using an equal
concentration of FBP without fluorogen as the reference.

[0032] FIGS. 16A, 16B and 16C are graphs depicting photobleaching curves
for FBPs. FIG. 16A is a graph of photobleaching curves for TO1-FBP and
EGFP displayed on yeast. JAR200 yeast strains displaying HL1.0.1-TO1 and
EGFP are immobilized on concanavalin-A treated 35 mm petri dishes with 14
mm optical microwell (MatTek Corp) and bleached in 2 ml modified PBS
using an Olympus IX50 inverted microscope equipped with a 100 W Hg lamp,
40×1.3NA oil objective and a Photometrics CoolSnap HQ camera with
HQ470/40 excitation and HQ500 LP emission filters (Chroma set #41018,
total irradiance at the specimen plane was measured at 30 mW (13.6
μW/μm2)). Each curve represents an average of scans of 8-12
individual cells. Fluorescence of EGFP is normalized (scaled down
˜2.5-fold) to match HL1.0.1-TO1 cells visualized with 375 nM
TO1-2p. FIG. 16B is a graph depicting the photobleaching lifetime of
yeast displayed TO1-FBP and EGFP. JAR200 yeast cells treated are bleached
on a Leica DMI 6000 B confocal microscope using 488 nm laser excitation
at 100% power and monitoring emission with a 500-600 nm window. Data from
individual cells are averaged and the EGFP signal is normalized (scaled
down ˜3-fold) to match initial HL1.0.1-TO1 fluorescence. Plotted
data points are displayed with a single exponential decay curve (Graphpad
Prism 4.0 software). Lifetimes are corrected by comparing excitation
intensities of these cells at 488 nm to the intensity at their excitation
maxima (EGFP at 502 nm, HL1.0.1-TO1 at 512 nm), determined on a Tecan
Safire2 plate reader. FIG. 16C is a graph depicting the photobleaching of
MG-FBP displayed on mammalian cells. NIH 3T3 cells stably expressing both
HL4-MG and HL1.1-TO1 simultaneously are isolated using FACS, and grown as
a layer on the optical window of 35 mm petri dishes. Bleaching
experiments are carried out in PBS w/ Ca and Mg using HQ620/60 excitation
and HQ665 LP emission filters (Chroma set #41024), total specimen plane
irradiance is measured at 30 mW.

[0033] FIG. 17 is a graph depicting the effect of fluorogens on yeast cell
growth. JAR200 cells are inoculated at ˜106 cells/ml into 35
ml SD+CAA medium in 125 ml baffled flasks and allowed to grow at
30° C. at 300 RPM for 2 hours prior to addition of fluorogens at
the indicated concentrations. One ml samples are removed at indicated
time points, and growth halted by addition of 75 μl of 300 mM
NaN3 prior to reading absorbance. Doubling time of about 1.9 hrs is
unchanged by most fluorogen treatments. For 500 nM MG-ester, the doubling
time is about 2.8 hrs; and for 500 nM MG, the doubling time is over 24
hrs.

[0034] FIG. 18A is a binding schematic for the FBP L5-MG. FIG. 18B is a
binding schematic for the FBP H8-MG.

[0035] FIG. 19A is a binding schematic for the FBP H6-MG. FIG. 19B is a
binding schematic for the FBP HL4-MG.

[0036] FIG. 20A is a binding schematic for the FBP HL1-TO1. FIG. 20B is a
binding schematic for the FBP HL1.0.1-TO1.

[0037] FIG. 21A is a binding schematic for the FBP HL1.1-TO1. FIG. 21B is
a binding schematic for the FBP HL7-MG.

[0038] FIG. 22A is a binding schematic for the FBP HL2-TO1. FIG. 22B is a
binding schematic for the FBP HL9-MG.

DETAILED DESCRIPTION

[0039] To provide an overall understanding, certain illustrative
embodiments will now be described; however, it will be understood by one
of ordinary skill in the art that the systems and methods described
herein can be adapted and modified to provide systems and methods for
other suitable applications and that other additions and modifications
can be made without departing from the scope of the systems and methods
described herein.

[0040] Unless otherwise specified, the illustrated embodiments can be
understood as providing exemplary features of varying detail of certain
embodiments, and therefore unless otherwise specified, features,
components, modules, and/or aspects of the illustrations can be combined,
separated, interchanged, and/or rearranged without departing from the
disclosed systems or methods.

[0041] 1. Introduction

[0042] For convenience, certain terms employed in the specification,
examples, and appended claims are collected here. Unless defined
otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art.

[0043] Other than in the examples herein, or unless otherwise expressly
specified, all of the numerical ranges, amounts, values and percentages,
such as those for amounts of materials, elemental contents, times and
temperatures of reaction, ratios of amounts, and others, in the following
portion of the specification and attached claims, may be read as if
prefaced by the word "about" even though the term "about" may not
expressly appear with the value, amount, or range. Accordingly, unless
indicated to the contrary, the numerical parameters set forth in the
following specification and claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to limit the
application of the doctrine of equivalents to the scope of the claims,
each numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary rounding
techniques.

[0044] Notwithstanding that the numerical ranges and parameters setting
forth the broad scope of the invention are approximations, the numerical
values set forth in the specific examples are reported as precisely as
possible. Any numerical value, however, inherently contains error
necessarily resulting from the standard deviation found in its underlying
respective testing measurements. Furthermore, when numerical ranges are
set forth herein, these ranges are inclusive of the recited range end
points (i.e., end points may be used). When percentages by weight are
used herein, the numerical values reported are relative to the total
weight.

[0045] Also, it should be understood that any numerical range recited
herein is intended to include all sub-ranges subsumed therein. For
example, a range of "1 to 10" is intended to include all sub-ranges
between (and including) the recited minimum value of 1 and the recited
maximum value of 10, that is, having a minimum value equal to or greater
than 1 and a maximum value of equal to or less than 10. The articles "a"
and "an" are used herein to refer to one or to more than one (i.e., to at
least one) of the grammatical object of the article. By way of example,
"an element" means one element or more than one element.

[0046] The term "amino acid" is intended to embrace all molecules, whether
natural or synthetic, which include both an amino functionality and an
acid functionality and capable of being included in a polymer of
naturally-occurring amino acids. Exemplary amino acids include
naturally-occurring amino acids; analogs, derivatives and congeners
thereof; amino acid analogs having variant side chains; and all
stereoisomers of any of any of the foregoing.

[0047] As used herein, the term "selectivity component" refers to a
molecule capable of interacting with a target molecule. Selectivity
components having limited cross-reactivity are generally preferred. In
certain embodiments, suitable selectivity components include, for
example, polypeptides, such as for example, antibodies, monoclonal
antibodies, or derivatives or analogs thereof, including without
limitation: Fv fragments, single chain Fv (scFv) fragments, Fab'
fragments, F(ab')2 fragments, single domain antibodies, camelized
antibodies and antibody fragments, humanized antibodies and antibody
fragments, and multivalent versions of the foregoing; multivalent binding
reagents including without limitation: monospecific or bispecific
antibodies, such as disulfide stabilized Fv fragments, scFv tandems
((scFv)2 fragments), diabodies, tribodies or tetrabodies, which
typically are covalently linked or otherwise stabilized (i. e., leucine
zipper or helix stabilized) scFv fragments; and other binding reagents
including, for example, aptamers, template imprinted materials (such as
those of U.S. Pat. No. 6,131,580), and organic or inorganic binding
elements. In exemplary embodiments, a selectivity component specifically
interacts with a single epitope. In other embodiments, a selectivity
component may interact with several structurally related epitopes.

[0048] The term "ligand" refers to a binding moiety for a specific target
molecule. The molecule can be a cognate receptor, a protein a small
molecule, a hapten, or any other relevant molecule.

[0049] The term "antibody" refers to an immunoglobulin, derivatives
thereof which maintain specific binding ability, and proteins having a
binding domain which is homologous or largely homologous to an
immunoglobulin binding domain. As such, the antibody operates as a ligand
for its cognate antigen, which can be virtually any molecule. Natural
antibodies comprise two heavy chains and two light chains and are
bi-valent. The interaction between the variable regions of heavy and
light chain forms a binding site capable of specifically binding an
antigen. The term "VH" refers to a heavy chain variable region of an
antibody. The term "VL" refers to a light chain variable region of
an antibody. Antibodies may be derived from natural sources, or partly or
wholly synthetically produced. An antibody may be monoclonal or
polyclonal. The antibody may be a member of any immunoglobulin class,
including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In
exemplary embodiments, antibodies used with the methods and compositions
described herein are derivatives of the IgG class.

[0050] The term "antibody fragment" refers to any derivative of an
antibody which is less than full-length. In exemplary embodiments, the
antibody fragment retains at least a significant portion of the
full-length antibody's specific binding ability. Examples of antibody
fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv,
dsFv, scFv, diabody, and Fd fragments. The antibody fragment may be
produced by any means. For instance, the antibody fragment may be
enzymatically or chemically produced by fragmentation of an intact
antibody, it may be recombinantly or partially synthetically produced.
The antibody fragment may optionally be a single chain antibody fragment.
Alternatively, the fragment may comprise multiple chains which are linked
together, for instance, by disulfide linkages. The fragment may also
optionally be a multimolecular complex. A functional antibody fragment
will typically comprise at least about 50 amino acids and more typically
will comprise at least about 200 amino acids.

[0051] The term "Fab" refers to an antibody fragment that is essentially
equivalent to that obtained by digestion of immunoglobulin (typically
IgG) with the enzyme papain. The heavy chain segment of the Fab fragment
is the Fd piece. Such fragments may be enzymatically or chemically
produced by fragmentation of an intact antibody, recombinantly produced
from a gene encoding the partial antibody sequence, or it may be wholly
or partially synthetically produced. Methods for preparing Fab fragments
are known in the art. See, for example, Tijssen, Practice and Theory of
Enzyme Immunoassays (Elsevier, Amsterdam, 1985).

[0052] The term "Fab" refers to an antibody fragment that is essentially
equivalent to that obtained by reduction of the disulfide bridge or
bridges joining the two heavy chain pieces in the F(ab')2 fragment.
Such fragments may be enzymatically or chemically produced by
fragmentation of an intact antibody, recombinantly produced from a gene
encoding the partial antibody sequence, or it may be wholly or partially
synthetically produced.

[0053] The term "F(ab')2" refers to an antibody fragment that is
essentially equivalent to a fragment obtained by digestion of an
immunoglobulin (typically IgG) with the enzyme pepsin at pH 4.0-4.5. Such
fragments may be enzymatically or chemically produced by fragmentation of
an intact antibody, recombinantly produced from a gene encoding the
partial antibody sequence, or it may be wholly or partially synthetically
produced.

[0054] The term "Fv" refers to an antibody fragment that consists of one
VH and one VL domain held together by noncovalent interactions.
The term "dsFv" is used herein to refer to an Fv with an engineered
intermolecular disulfide bond to stabilize the VH-VL pair.
Methods for preparing Fv fragments are known in the art. See, for
example, Moore et al., U.S. Pat. No. 4,462,334; Hochman et al.,
Biochemistry 12: 1130 (1973); Sharon et al., Biochemistry 15: 1591(1976);
and Ehrlich et al., U.S. Pat. No. 4,355,023.

[0055] The terms "single-chain Fvs" and "scFvs" refers to recombinant
antibody fragments consisting of only the variable light chain (VL)
and variable heavy chain (VH) covalently connected to one another by
a polypeptide linker. Either VL or VH may be the
NH2-terminal domain. The polypeptide linker may be of variable
length and composition so long as the two variable domains are bridged
without serious steric interference. In exemplary embodiments, the
linkers are comprised primarily of stretches of glycine and serine
residues with some glutamic acid or lysine residues interspersed for
solubility. Methods for preparing scFvs are known in the art. See, for
example, PCT/US/87/02208 and U.S. Pat. No. 4,704,692.

[0056] The term "single domain antibody" or "Fd" refers to an antibody
fragment comprising a VH domain that interacts with a given antigen.
An Fd does not contain a VL domain, but may contain other antigen
binding domains known to exist in antibodies, for example, the kappa and
lambda domains. In certain embodiments, the Fd comprises only the FL
component. Methods for preparing Fds are known in the art. See, for
example, Ward et al., Nature 341:644-646 (1989) and EP 0368684 A1.

[0057] The term "single chain antibody" refers to an antibody fragment
that comprises variable regions of the light and heavy chains joined by a
flexible linker moiety. Methods for preparing single chain antibodies are
known in the art. See, for example, U.S. Pat. No. 4,946,778 to Ladner et
al.

[0058] The term "diabodies" refers to dimeric scFvs. The components of
diabodies typically have shorter peptide linkers than most scFvs and they
show a preference for associating as dimers. The term diabody is intended
to encompass both bivalent (i.e., a dimer of two scFvs having the same
specificity) and bispecific (i.e., a dimer of two scFvs having different
specificities) molecules. Methods for preparing diabodies are known in
the art. See, for example, EP 404097 and WO93/11161.

[0059] The term "triabody" refers to trivalent constructs comprising 3
scFv's, and thus comprising 3 variable domains (see, e.g., Iliades et
al., FEBS Lett. 409(3):43741 (1997)). Triabodies is meant to include
molecules that comprise 3 variable domains having the same specificity,
or 3 variable domains wherein two or more of the variable domains have
different specificities.

[0060] The term "tetrabody" refers to engineered antibody constructs
comprising 4 variable domains (see, e.g., Pack et al., J Mol Biol.
246(1): 28-34 (1995) and Coloma & Morrison, Nat Biotechnol. 15(2): 159-63
(1997)). Tetrabodies is meant to include molecules that comprise 4
variable domains having the same specificity, or 4 variable domains
wherein two or more of the variable domains have different specificities.

[0061] The term "camelized antibody" refers to an antibody or variant
thereof that has been modified to increase its solubility and/or reduce
aggregation or precipitation. For example; camelids produce heavy-chain
antibodies consisting only of a pair of heavy chains wherein the antigen
binding site comprises the N-terminal variable region or VHH
(variable domain of a heavy chain antibody). The VHH domain
comprises an increased number of hydrophilic amino acid residues that
enhance the solubility of a VHH domain as compared to a VH
region from non-camelid antibodies. Camelization of an antibody or
variant thereof involves replacing one or more amino acid residues of a
non-camelid antibody with corresponding amino residues from a camelid
antibody.

[0062] As used herein, the term "epitope" refers to a physical structure
on a molecule that interacts with a selectivity component, such as an
antibody. In exemplary embodiments, epitope refers to a desired region on
a target molecule that specifically interacts with a selectivity
component.

[0063] "Interact" is meant to include detectable interactions between
molecules, such as may be detected using, for example, a hybridization
assay. Interact also includes "binding" interactions between molecules.
Interactions may be, for example, protein-protein, protein-nucleic acid,
protein-small molecule or small molecule-nucleic acid, and includes for
example, antibody-antigen binding, receptor-ligand binding,
hybridization, and other forms of binding. In certain embodiments, an
interaction between a ligand and a specific target will lead to the
formation of a complex, wherein the ligand and the target are unlikely to
dissociate. Such affinity for a ligand and its target can be defined by
the dissociation constant (Kd) as known in the art. A complex may
include a ligand for a specific dye and is referred to herein as a
"ligand-dye" complex.

[0064] The term "immunogen" traditionally refers to compounds that are
used to elicit an immune response in an animal, and is used as such
herein. However, many techniques used to produce a desired selectivity
component, such as the phage display and aptamer methods described below,
do not rely wholly, or even in part, on animal immunizations.
Nevertheless, these methods use compounds containing an "epitope," as
defined above, to select for and clonally expand a population of
selectivity components specific to the "epitope." These in vitro methods
mimic the selection and clonal expansion of immune cells in vivo, and,
therefore, the compounds containing the "epitope" that is used to
clonally expand a desired population of phage, aptamers and the like in
vitro are embraced within the definition of "immunogens."

[0065] Similarly, the terms "hapten" and "carrier" have specific meaning
in relation to the immunization of animals, that is, a "hapten" is a
small molecule that contains an epitope, but is incapable as serving as
an immunogen alone. Therefore, to elicit an immune response to the
hapten, the hapten is conjugated with a larger carrier, such as bovine
serum albumin or keyhole limpet hemocyanin, to produce an immunogen. A
preferred immune response would recognize the epitope on the hapten, but
not on the carrier. As used herein in connection with the immunization of
animals, the terms "hapten" and "carrier" take on their classical
definition. However, in the in vitro methods described herein for
preparing the desired binding reagents, traditional "haptens" and
"carriers" typically have their counterpart in epitope-containing
compounds affixed to suitable substrates or surfaces, such as beads and
tissue culture plates.

[0066] The term "aptamer" refers to a nucleic acid molecule that may
selectively interact with a non-oligonucleotide molecule or group of
molecules. In various embodiments, aptamers may include single-stranded,
partially single-stranded, partially double-stranded or double-stranded
nucleic acid sequences; sequences comprising nucleotides,
ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified
nucleotides and nucleotides comprising backbone modifications,
branchpoints and nonnucleotide residues, groups or bridges; synthetic
RNA, DNA and chimeric nucleotides, hybrids, duplexes, heteroduplexes; and
any ribonucleotide, deoxyribonucleotide or chimeric counterpart thereof
and/or corresponding complementary sequence. In certain embodiments,
aptamers may include promoter or primer annealing sequences that may be
used to amplify, transcribe or replicate all or part of the aptamer.

[0067] As used herein, the term "reporter molecule" refers to a molecule
suitable for detection, such as, for example, spectroscopic detection.
Examples of reporter molecules include, but are not limited to, the
following: fluorescent labels, enzymatic labels, biotinyl groups, and
predetermined polypeptide epitopes recognized by a secondary reporter
(e.g., leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding domains, epitope tags). Examples and use of
such reporter molecules are described in more detail below. In some
embodiments, reporter molecules are attached by spacer arms of various
lengths to reduce potential steric hindrance. Reporter molecules may be
incorporated into or attached (including covalent and non-covalent
attachment) to a molecule, such as a selectivity component. Various
methods of labeling polypeptides are known in the art and may be used.

[0068] As used herein, the term "sensor dye" refers to a reporter molecule
that exhibits an increase, decrease or modification of signal in response
to a change in the environment. In exemplary embodiments, the sensor dye
is a fluorescent molecule that is responsive to changes in polarity
and/or mobility of the dye, as well as, the changes microenvironment pH
and/or viscosity, or combinations thereof.

[0069] A "fusion protein" or "fusion polypeptide" refers to a chimeric
protein as that term is known in the art and may be constructed using
methods known in the art. In many examples of fusion proteins, there are
two different polypeptide sequences, and in certain cases, there may be
more. The sequences may be linked in frame. A fusion protein may include
a domain which is found (albeit in a different protein) in an organism
which also expresses the first protein, or it may be an "interspecies",
"intergenic", etc. fusion expressed by different kinds of organisms. In
various embodiments, the fusion polypeptide may comprise one or more
amino acid sequences linked to a first polypeptide. In the case where
more than one amino acid sequence is fused to a first polypeptide, the
fusion sequences may be multiple copies of the same sequence, or
alternatively, may be different amino acid sequences. The fusion
polypeptides may be fused to the N-terminus, the C-terminus, or the N-
and C-terminus of the first polypeptide. Exemplary fusion proteins
include polypeptides comprising a glutathione S-transferase tag
(GST-tag), histidine tag (His-tag), an immunoglobulin domain or an
immunoglobulin binding domain.

[0070] As used herein, the term "array" refers to a set of selectivity
components immobilized onto one or more substrates so that each
selectivity component is at a known location. In an exemplary embodiment,
a set of selectivity components is immobilized onto a surface in a
spatially addressable manner so that each individual selectivity
component is located at a different and identifiable location on the
substrate.

[0071] The term "chemical handle" refers to a component that may be
attached to a biosensor as described herein so as to facilitate its
isolation, immobilization, identification, or detection and/or which
increases its solubility. Suitable chemical handles include, for example,
a polypeptide, a polynucleotide, a carbohydrate, a polymer, or a chemical
moiety and combinations or variants thereof.

[0072] The term "conserved residue" refers to an amino acid that is a
member of a group of amino acids having certain common properties. The
term "conservative amino acid substitution" refers to the substitution
(conceptually or otherwise) of an amino acid from one such group with a
different amino acid from the same group. A functional way to define
common properties between individual amino acids is to analyze the
normalized frequencies of amino acid changes between corresponding
proteins of homologous organisms (Schulz, G. I,. and R. H. Schirmer,
Principles of Protein Structure, Springer-Verlag). According to such
analyses, groups of amino acids may be defined where amino acids within a
group exchange preferentially with each other, and therefore resemble
each other most in their impact on the overall protein structure (Schulz,
Schirmer, Principles of Protein Structure, Springer-Verlag). One example
of a set of amino acid groups defined in this manner include: (i) a
charged group, consisting of Glu and Asp, Lys, Arg and His, (ii) a
positively-charged group, consisting of Lys, Arg and His, (iii) a
negatively-charged group, consisting of Glu and Asp, (iv) an aromatic
group, consisting of Phe, Tyr and Trp, (v) a nitrogen ring group,
consisting of His and Trp, (vi) a large aliphatic nonpolar group,
consisting of Val, Leu and Ile, (vii) a slightly-polar group, consisting
of Met and Cys, (viii) a small-residue group, consisting of Ser, Thr,
Asp, Asn, Gly, Ala, Glu, Gln and Pro, (ix) an aliphatic group consisting
of Val, Leu, Ile, Met and Cys, and (x) a small hydroxyl group consisting
of Ser and Thr.

[0073] "Isolated", with respect to nucleic acids, such as DNA or RNA,
refers to molecules separated from other DNAs, or RNAs, respectively,
that are present in the natural source of the macromolecule. Isolated
also refers to a nucleic acid or peptide that is substantially free of
cellular material, viral material, or culture medium when produced by
recombinant DNA techniques, or chemical precursors or other chemicals
when chemically synthesized. Moreover, an "isolated nucleic acid" is
meant to include nucleic acid fragments which are not naturally occurring
as fragments and would not be found in the natural state. "Isolated" also
refers to polypeptides which are isolated from other cellular proteins
and is meant to encompass both purified and recombinant polypeptides.

[0074] The term "mammal" is known in the art, and exemplary mammals
include humans, primates, bovines, porcines, canines, felines, and
rodents (e.g., mice and rats).

[0075] The term "microenvironment" refers to localized conditions within a
larger area. For example, association of two molecules within a solution
may alter the local conditions surrounding the associating molecules
without affecting the overall conditions within the solution.

[0076] The term "nucleic acid" refers to a polymeric form of nucleotides,
either ribonucleotides or deoxynucleotides or a modified form of either
type of nucleotide. The terms should also be understood to include, as
equivalents, analogs of either RNA or DNA made from nucleotide analogs,
and, as applicable to the embodiment being described, single-stranded
(such as sense or antisense) and double-stranded polynucleotides.

[0077] The term "polypeptide", and the terms "protein" and "peptide" which
are used interchangeably herein, refers to a polymer of amino acids.

[0078] The terms "polypeptide fragment" or "fragment", when used in
regards to a reference polypeptide, refers to a polypeptide in which
amino acid residues are deleted as compared to the reference polypeptide
itself, but where the remaining amino acid sequence is usually identical
to the corresponding positions in the reference polypeptide. Such
deletions may occur at the amino-terminus or carboxy-terminus of the
reference polypeptide, or alternatively both. Fragments typically are at
least 5, 10, 20, 50, 100, 500 or more amino acids long. A fragment can
retain one or more of the biological activities of the reference
polypeptide.

[0079] The term "sequence homology" refers to the proportion of base
matches between two nucleic acid sequences or the proportion of amino
acid matches between two amino acid sequences. When sequence homology is
expressed as a percentage, e.g., 50%, the percentage denotes the
proportion of matches over the length of sequence from a desired sequence
(e.g., SEQ. ID NO: 1) that is compared to some other sequence. Gaps (in
either of the two sequences) are permitted to maximize matching; gap
lengths of 15 bases or less are usually used, 6 bases or less are used
more frequently, with 2 bases or less used even more frequently. The term
"sequence identity" means that sequences are identical (i.e., on a
nucleotide-by-nucleotide basis for nucleic acids or amino acid-by-amino
acid basis for polypeptides) over a window of comparison. The term
"percentage of sequence identity" is calculated by comparing two
optimally aligned sequences over the comparison window, determining the
number of positions at which the identical amino acids occurs in both
sequences to yield the number of matched positions, dividing the number
of matched positions by the total number of positions in the comparison
window, and multiplying the result by 100 to yield the percentage of
sequence identity. Methods to calculate sequence identity are known to
those of skill in the art.

[0080] 2. Biosensors

[0081] Provided are biosensors, compositions comprising biosensors, and
methods of using biosensors in living cells and organisms. The biosensors
are able to be selectively targeted to certain regions or structures
within a cell. The biosensors may provide a signal when the biosensor is
targeted and/or in response to a property of the cell or organism such as
membrane potential, ion concentration or enzyme activity.

[0082] In general, the biosensors comprise at least two components; (1) a
selectivity component capable of interacting with a target molecule of
interest and a (2) reporter molecule that produces a detectable change in
signal upon interaction of the selectivity component with the target
molecule. The reporter molecule may be covalently linked to the
selectivity component, or it may be able to noncovalently interact with
the selectivity component.

[0083] In various embodiments, the reporter molecule is responsive to
environmental changes, including for example, pH sensitive molecules,
restriction sensitive molecules, polarity sensitive molecules, and
mobility sensitive molecules. The reporter molecule may be either
fluorescent or chemiluminescent. In certain embodiments, the reporter
molecule may interact with the selectivity component proximal to a region
that binds to the target molecule. In an exemplary embodiment, the
reporter molecule is covalently attached to the selectivity component
proximal to a region that binds to the target molecule, optionally
through an engineered reactive site. The biosensor may respond to changes
in the concentration of the target molecule and may be useful for
monitoring the concentration of a target molecule over time.

[0084] In certain embodiments, the biosensor may comprise two or more
reporter molecules, which may be the same or different reporter
molecules. The reporter molecule may be detectable by a variety of
methods, including, for example, a fluorescent spectrometer, filter
fluorometer, microarray reader, optical fiber sensor reader,
epifluorescence microscope, confocal laser scanning microscope, two
photon excitation microscope, or a flow cytometer.

[0085] In certain embodiments, methods for preparing the biosensors
include generating selectivity components with an engineered reporter
molecule binding site using biological selection methods. The reporter
molecule binding site may be engineered to customize any of a number of
properties, for example, for optimal binding affinity to the reporter
molecule, to enhance or otherwise change or tune the signal from the
reporter molecule when it binds the selectivity component, to provide a
reactive site in the reporter molecule binding site so that the reporter
molecule may covalently associate with the selectivity component upon
binding in the binding site, or to modulate or perturb the activity of
the selectivity component when the reporter molecule binds to it.

[0086] Accordingly, in certain embodiments, methods for generating a
biosensor may comprise producing the selectivity component by genetic
selection, genetic engineering or a combination of genetic selection and
genetic engineering, so as to produce an engineered selectivity
component. Methods for producing the engineered selectivity components
are described further below.

[0087] In other embodiments, particularly where the biosensor is produced
from an endogenous source rather than expressed in the cell or tissue to
be analyzed, the biosensor may further comprise a chemical handle. The
chemical handle may be used to facilitate isolation, immobilization,
identification, or detection of the biosensors and/or which increases the
solubility of the biosensors.

[0089] In another embodiment, the application provides a composition
comprising one or more biosensors. The composition may comprise a
pharmaceutically acceptable carrier. The biosensors of the composition
may be specific for different target molecules, and may be associated
with the same or different reporter molecules.

[0090] In another embodiment, two or more biosensors may be immobilized
onto a substrate at spatially addressable locations. The biosensors may
be specific for different target molecules and may be associated with the
same or different reporter molecules.

[0091] In another aspect, the application provides a method for detecting
at least one target molecule comprising providing at least one biosensor
comprising a selectivity component and a reporter molecule and detecting
the signal of the reporter molecule, wherein interaction of the biosensor
with the target molecule produces a detectable change in the signal of
the reporter molecule. In various other aspects, the biosensors of the
invention may be used for the detection of environmental pollutants,
hazardous substances, food contaminants, and biological and/or chemical
warfare agents.

[0092] In various embodiments, the biosensors of the invention may be used
to detect target molecules, including, for example, cells, microorganisms
(bacteria, fungi and viruses), polypeptides, nucleic acids, hormones,
cytokines, drug molecules, carbohydrates, pesticides, dyes, amino acids,
small organic molecules and small inorganic molecules.

[0093] Biosensors may be used for the detection of target molecules both
in vivo and in vitro. In certain embodiments, the biosensor may be
injected or implanted into a patient and the signal of the reporter
molecule is detected externally. In one exemplary embodiment, the
biosensors of the application may be used for the detection of
intracellular targets. In another exemplary embodiment, the biosensors of
the application may be attached to a fiber optic probe to facilitate
position of the biosensor within a sample and readout from the biosensor
through the optical fiber.

[0094] In still other embodiments, the biosensor may be expressed directly
into the cell, tissue or subject to be analyzed. Using molecular biology
methods, a vector comprising at least a gene encoding a selectivity
component is constructed and inserted into the host, resulting in
expression of the selectivity component, as described in more detail
below.

[0095] Various, more detailed embodiments of and methods for producing the
selectivity component and reporter molecule components are also further
described below.

[0098] Exemplary target molecules include, for example, molecules involved
in tissue differentiation and/or growth, cellular communication, cell
division, cell motility, and other cellular functions that take place
within or between cells, including regulatory molecules such as growth
factors, cytokines, morphogenetic factors, neurotransmitters, and the
like. In certain embodiments, target molecules may be bone morphogenic
protein, insulin-like growth factor (IGF), and/or members of the hedgehog
and Wnt polypeptide families.

[0101] In certain embodiments, the selectivity component may be an
antibody or an antibody fragment. For example, selectivity components may
be monoclonal antibodies, or derivatives or analogs thereof, including
without limitation: Fv fragments, single chain Fv (scFv) fragments, Fab'
fragments, F(ab')2 fragments, single domain antibodies, camelized
antibodies and antibody fragments, humanized antibodies and antibody
fragments, and multivalent versions of the foregoing; multivalent
selectivity components including without limitation: monospecific or
bispecific antibodies, such as disulfide stabilized Fv fragments, scFv
tandems ((scFv)2 fragments), diabodies, tribodies or tetrabodies,
which typically are covalently linked or otherwise stabilized (i.e.,
leucine zipper or helix stabilized) scFv fragments; receptor molecules
which naturally interact with a desired target molecule.

[0102] In one embodiment, the selectivity component may be an antibody.
Preparation of antibodies may be accomplished by any number of well-known
methods for generating monoclonal antibodies. These methods typically
include the step of immunization of animals, typically mice; with a
desired immunogen (e.g., a desired target molecule- or fragment thereof).
Once the mice have been immunized, and preferably boosted one or more
times with the desired immunogen(s), monoclonal antibody-producing
hybridomas may be prepared and screened according to well known methods
(see, for example, Kuby, Janis, IMMUNOLOGY, Third Edition, pp. 131-139,
W.H. Freeman & Co. (1997), for a general overview of monoclonal antibody
production, that portion of which is incorporated herein by reference).

[0103] Over the past several decades, antibody production has become
extremely robust. In vitro methods that combine antibody recognition and
phage display techniques allow one to amplify and select antibodies with
very specific binding capabilities. See, for example, Holt, L. J. et al.,
"The Use of Recombinant Antibodies in Proteomics," Current Opinion in
Biotechnology 2000, 11:445-449, incorporated herein by reference. These
methods typically are much less cumbersome than preparation of hybridomas
by traditional monoclonal antibody preparation methods. Binding epitopes
may range in size from small organic compounds such as bromo uridine and
phosphotyrosine to oligopeptides on the order of 7-9 amino acids in
length.

[0104] In another embodiment, the selectivity component may be an antibody
fragment. Preparation of antibody fragments may be accomplished by any
number of well-known methods. In one embodiment, phage display technology
may be used to generate antibody fragment selectivity components that are
specific for a desired target molecule, including, for example, Fab
fragments, Fv's with an engineered intermolecular disulfide bond to
stabilize the VH-VL pair, scFvs, or diabody fragments.

[0105] In certain embodiments, the selectivity component comprises a
polypeptide sequence having at least about 85%, at least about 90%, at
least about 95%, about 96%, about 97%, about 98%, about 99% or about 100%
sequence identity to the polypeptide sequence of SEQ ID NO: 2 (FIG. 1B).
Vectors to produce the selectivity component may be prepared as described
below with the nucleic acid encoding the polypeptide of SEQ ID NO:2 and
its homologs (for example, SEQ ID NO: 1 in FIG. 1A), and used to
transfect host cells as described further below.

[0106] As an example, production of scFv antibody fragments using phage
display is described below. However, scFv antibody fragments for use in
the selectivity components may be generated by any method known in the
art for doing so, including genetic selection methods from a library of
yeast cells (see Boder and Wittrup (2000) Meth. Enzymol. 328:430-33;
Boder, et al. (2000) Proc. Natl. Acad. Sci USA 97:10701-5; and Swers, et
al. (2004) Nucl. Acids. Res. 32:e36).

[0107] For phage display, an immune response to a selected immunogen is
elicited in an animal (such as a mouse, rabbit, goat or other animal) and
the response is boosted to expand the immunogen-specific B-cell
population. Messenger RNA is isolated from those B-cells, or optionally a
monoclonal or polyclonal hybridoma population. The mRNA is
reverse-transcribed by known methods using either a poly-A primer or
murine immunoglobulin-specific primer(s), typically specific to sequences
adjacent to the desired VH and VL chains, to yield cDNA. The
desired VH and A chains are amplified by polymerase chain reaction
(PCR) typically using VH and A specific primer sets, and are ligated
together, separated by a linker. VH and VL specific primer sets
are commercially available, for instance from Stratagene, Inc. of La
Jolla, Calif. Assembled VH-linker-VL product (encoding an scFv
fragment) is selected for and amplified by PCR. Restriction sites are
introduced into the ends of the VH-linker-VL product by PCR
with primers including restriction sites and the scFv fragment is
inserted into a suitable expression vector (typically a plasmid) for
phage display. Other fragments, such as an Fab' fragment, may be cloned
into phage display vectors for surface expression on phage particles. The
phage may be any phage, such as lambda, but typically is a filamentous
phage, such as fd and M 13, typically M13.

[0108] In phage display vectors, the VH-linker-VL sequence is
cloned into a phage surface protein (for M 13, the surface proteins g3p
(pHI) or g8p, most typically g3p). Phage display systems also include
phagemid systems, which are based on a phagemid plasmid vector containing
the phage surface protein genes (for example, g3p and g8p of M13) and the
phage origin of replication. To produce phage particles, cells containing
the phagemid are rescued with helper phage providing the remaining
proteins needed for the generation of phage. Only the phagemid vector is
packaged in the resulting phage particles because replication of the
phagemid is grossly favored over replication of the helper phage DNA.
Phagemid packaging systems for production of antibodies are commercially
available. One example of a commercially available phagemid packaging
system that also permits production of soluble ScFv fragments in bacteria
cells is the Recombinant Phage Antibody System (R. PAS), commercially
available from Amersham Pharmacia Biotech, Inc. of Piscataway, N.J. and
the pSKAN Phagemid Display System, commercially available from MoBiTec,
LLC of Marco Island, Fla. Phage display systems, their construction and
screening methods are described in detail in, among others, U.S. Pat.
Nos. 5,702,892, 5,750,373, 5,821,047 and 6,127, 132, each of which are
incorporated herein by reference in their entirety.

[0109] Typically, once phage are produced that display a desired antibody
fragment, epitope specific phage are selected by their affinity for the
desired immunogen and, optionally, their lack be used for physically
separating immunogen-binding phage from non-binding phage. Typically the
immunogen is fixed to a surface and the phage are contacted with the
surface. Non-binding phage are washed away while binding phage remain
bound. Bound phage are later eluted and are used to re-infect cells to
amplify the selected species. A number of rounds of affinity selection
typically are used, often increasingly higher stringency washes, to
amplify immunogen binding phage of increasing affinity. Negative
selection techniques also may be used to select for lack of binding to a
desired target. In that case, un-bound (washed) phage are amplified.

[0110] Although it is preferred to use spleen cells and/or B-lymphocytes
from animals preimmunized with a desired immunogen as a source of cDNA
from which the sequences of the VH and VL chains are amplified
by RT-PCR, naive (un-immunized with the target immunogen) splenocytes
and/or B-cells may be used as a source of cDNA to produce a polyclonal
set of VH and VL chains that are selected in vitro by affinity,
typically by the above-described phage display (phagemid) method. When
naive B-cells are used, during affinity selection, the washing of the
first selection step typically is of very high stringency so as to avoid
loss of any single clone that may be present in very low copy number in
the polyclonal phage library. By this naive method, B-cells may be
obtained from any polyclonal source, B-cell or splenocyte cDNA libraries
also are a source of cDNA from which the VH and VL chains may
be amplified. For example, suitable murine and human B-cell, lymphocyte
and splenocyte cDNA libraries are commercially available from Stratagene,
Inc. and from Clontech Laboratories, Inc. of Palo Alto, Calif. Phagemid
antibody libraries and related screening services are provided
commercially by Cambridge Antibody Technology of the U.K. or MorphoSys
USA, Inc., of Charlotte, N.C.

[0111] The selectivity components do not have to originate from biological
sources, such as from naive or immunized immune cells of animals or
humans. The selectivity components may be screened from a combinatorial
library of synthetic peptides. One such method is described in U.S. Pat.
No. 5,948,635, incorporated herein by reference, which described the
production of phagemid libraries having random amino acid insertions in
the pIII gene of M13. These phage may be clonally amplified by affinity
selection as described above.

[0112] Panning in a culture dish or flask is one way to physically
separate binding phage from non-binding phage. Panning may be carried out
in 96 well plates in which desired immunogen structures have been
immobilized. Functionalized 96 well plates, typically used as ELISA
plates, may be purchased from Pierce of Rockwell, Ill. Polypeptides
immunogens may be synthesized directly on NH2 or COOH functionalized
plates in an N-terminal to C-terminal direction. Other affinity methods
for isolating phage having a desired specificity include affixing the
immunogen to beads. The beads may be placed in a column and phage may be
bound to the column, washed and eluted according to standard procedures.
Alternatively, the beads may be magnetic so as to permit magnetic
separation of the binding particles from the non-binding particles. The
immunogen also may be affixed to a porous membrane or matrix, permitting
easy washing and elution of the binding phage.

[0113] In certain embodiments, it may be desirable to increase the
specificity of the selectivity component for a given target molecule or
reporter molecule using a negative selection step in the affinity
selection process. For example, selectivity component displaying phage
may be contacted with a surface functionalized with immunogens distinct
from the target molecule or reporter molecule. Phage are washed from the
surface and non-binding phage are grown to clonally expand the population
of non-binding phage thereby de-selecting phage that are not specific for
the desired target molecule. In certain embodiments, random synthetic
peptides may be used in the negative selection step. In other
embodiments, one or more immunogens having structural similarity to the
target molecule or reporter molecule may be used in the negative
selection step. For example, for a target molecule comprising a
polypeptide, structurally similar immunogens may be polypeptides having
conservative amino acid substitutions, including but not limited to the
conservative substitution groups such as: (i) a charged group, consisting
of Glu and Asp, Lys, Arg and His, (ii) a positively-charged group,
consisting of Lys, Arg and His, (iii) a negatively-charged group,
consisting of Glu and Asp, (iv) an aromatic group, consisting of Phe, Tyr
and Trp, (v) a nitrogen ring group, consisting of His and Trp, (vi) a
large aliphatic nonpolar group, consisting of Val, Leu and Ile, (vii) a
slightly polar group, consisting of Met and Cys, (viii) a small-residue
group, consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln and Pro, (ix)
an aliphatic group consisting of Val, Leu, Ile, Met and Cys, and (x) a
small hydroxyl group consisting of Ser and Thr. Conservative
substitutions also may be determined by one or more methods, such as
those used by the BLAST (Basic Local Alignment Search Tool) algorithm,
such as a BLOSUM Substitution Scoring Matrix; such as the BLOSUM 62
matrix, and the like. A functional way to define common properties
between individual amino acids is to analyze the normalized frequencies
of amino acid changes between corresponding proteins of homologous
organisms (Schulz, G. E. and R H. Schirmer, Principles of Protein
Structure, Springer-Verlag).

[0114] Screening of selectivity components will best be accomplished by
high throughput parallel selection, as described in Holt et al.
Alternatively, high throughput parallel selection may be conducted by
commercial entities, such as by Cambridge Antibody Technologies or
MorphoSys USA, Inc.

[0115] Alternatively, selection of a desired selectivity
component-displaying phage may be carried out using the following method:

[0116] Step 1: Affinity purify phage under low stringency conditions for
their ability to bind to an immunogen fixed to a solid support (for
instance, beads in a column).

[0117] Step 2: Elute the bound phage and grow the eluted phage. Steps I
and 2 may be repeated with more stringent washes in Step 1.

[0118] Step 3: Absorb the phage under moderate stringency with a given
protein mixture digested with a proteolytic agent of interest. Wash away
the unbound phage with a moderately stringent wash and grow the washed
phage. Step 3 may be repeated with less stringent washes.

[0119] Step 4: Affinity purify phage under high stringency for their
ability to bind to the immunogen fixed to a solid support. Elute the
bound phage and grow the eluted phage.

[0120] Step 5: Plate the phage to select single plaques. Independently
grow phage selected from each plaque and confirm the specificity to the
desired immunogen.

[0121] This is a general-guideline for the clonal expansion of
immunogen-specific selectivity components. Additional steps of varying
stringency may be added at any stage to optimize the selection process,
or steps may be omitted or re-ordered. One or more steps may be added
where the phage population is selected for its inability to bind to other
immunogens by absorption of the phage population with those other
immunogens and amplification of the unbound phage population. That step
may be performed at any stage, but typically would be performed after
step 4.

[0122] In certain embodiments, it may be desirable to mutate the binding
region of the selectivity component and select for selectivity components
with superior binding characteristics as compared to the un-mutated
selectivity component. This may be accomplished by any standard
mutagenesis technique, such as by PCR with Taq polymerase under
conditions that cause errors. In such a case, the PCR:primers could be
used to amplify scFv-encoding sequences of phagemid plasmids under
conditions that would cause mutations. The PCR product may then be cloned
into a phagemid vector and screened for the desired specificity, as
described above.

[0123] In other embodiments, the selectivity components may be modified to
make them more resistant to cleavage by proteases. For example, the
stability of the selectivity components of the present invention that
comprise polypeptides may be increased by substituting one or more of the
naturally occurring amino acids in the (L) configuration with D-amino
acids. In various embodiments, at least 1%, 5%, 10%, 20%, 50%, 80%, 90%
or 100% of the amino acid residues of the selectivity components may be
of the D configuration. The switch from L to D amino acids neutralizes
the digestion capabilities of many of the ubiquitous peptidases found in
the digestive tract. Alternatively, enhanced stability of the selectivity
components of the invention may be achieved by the introduction of
modifications of the traditional peptide linkages. For example,
the-introduction of a cyclic ring within the polypeptide backbone may
confer enhanced stability in order to circumvent the effect of many
proteolytic enzymes known to digest polypeptides in the stomach or other
digestive organs and in serum. In still other embodiments, enhanced
stability of the selectivity components may be achieved by intercalating
one or more dextrorotatory amino acids (such as, dextrorotatory
phenylalanine or dextrorotatory tryptophan) between the amino acids of
the selectivity component. In exemplary embodiments, such modifications
increase the protease resistance of the selectivity components without
affecting their activity or specificity of interaction with a desired
target molecule or reporter molecule.

[0124] In certain embodiments, the antibodies or variants thereof, may be
modified to make them less immunogenic when administered to a subject.
For example, if the subject is human, the antibody may be "humanized";
where the complimentarity determining region(s) of the hybridoma-derived
antibody has been transplanted into a human monoclonal antibody, for
example as described in Jones, P. et al. (1986), Nature 321, 522-525,
Tempest et al. (1991) Biotechnology 9, 266-273, and U.S. Pat. No.
6,407,213. Also, transgenic mice, or other mammals, may be used to
express humanized antibodies. Such humanization may be partial or
complete.

[0125] In another embodiment, the selectivity component is a Fab fragment.
Fab antibody fragments may be obtained by proteolysis of an
immunoglobulin molecule using the protease papain. Papain digestion
yields two identical antigen-binding fragments, termed "Fab fragments",
each with a single antigen-binding site, and a residual "Fc fragment". In
an exemplary embodiment, papain is first activated by reducing the
sulfhydryl group in the active site with cysteine, mercaptoethanol or
dithiothreitol. Heavy metals in the stock enzyme may be removed by
chelation with EDTA (2 mM) to ensure maximum enzyme activity. Enzyme and
substrate' are normally mixed together in the ratio of 1:100 by weight.
After incubation, the reaction can be stopped by irreversible alkylation
of the thiol group with iodoacetamide or simply by dialysis. The
completeness of the digestion should be monitored by SDS-PAGE and the
various fractions separated by protein A-Sepharose or ion exchange
chromatography.

[0126] In still another embodiment, the selectivity component is a
F(ab')2 fragment. F(ab')2 antibody fragments may be prepared
from IgG molecules using limited proteolysis with the enzyme pepsin.
Exemplary conditions for pepsin proteolysis are 100 times antibody excess
w/w in acetate buffer at pH 4.5 and 37° C. Pepsin treatment of
intact immunoglobulin molecules yields an F(ab')2 fragment that has
two antigen-combining sites and is still capable of crosslinking antigen.
Fab' antibody fragments may be obtained by reducing F(ab')2
fragments using 2-mercaptoethylamine. The Fab' fragments may be separated
from unsplit F(ab')2 fragments and concentrated by application to a
Sephadex G-25 column (MT=46,000-58,000). In other embodiments, the
selectivity component may be a non-antibody receptor molecule, including,
for example, receptors which naturally recognize a desired target
molecule, receptors which have been modified to increase their
specificity of interaction with a target molecule, receptor molecules
which have been modified to interact with a desired target molecule not
naturally recognized by the receptor, and fragments of such receptor
molecules (see, e.g., Skerra, J. Molecular Recognition 13: 167-187
(2000)).

[0127] In other embodiments, the selectivity component may be a network or
pathway protein such as an enzyme, for example, a phosphatase or kinase.
Such proteins may be mutated to create a binding site for a reporter
and/or target molecule. For example, a method of creating a biosensor
from network and pathway proteins in cells and tissues may comprise
mutating a specific region on the selected protein to create a binding
site for a reporter or target molecule. The region selected for mutation
may be randomly or partially randomly mutated by creating mutations in
selected regions of the gene that codes for the protein that is to be
converted into a selectivity component. The gene with the mutated
region(s) may be incorporated by transfection into a system capable of
expressing the protein in a way that allows reporter molecule (or target
molecule) binding and fluorescence sensitivity to the activity (if a
reporter molecule) to be assayed. For example, the DNA with the mutated
region may be transfected into yeast cells that are able to express many
copies of the mutated protein molecules on the cell surface (see Boder
and Wittrup (2000) Meth. Enzymol. 328:430-33; Boder, et al. (2000) Proc.
Natl. Acad. Sci USA 97:10701-5; and Swers, et al. (2004) Nucl. Acids.
Res. 32:e36). By isolating and identifying by selection methods the
genetic sequence of the particular protein within the mutated population
that functions optimally as a selectivity component. For example,
reporter molecule binding mutants may be detected and selected using
magnetic bead separation and by flow cytometry or image cytometry.
Mutants that show a particular fluorescence signal change from bound
reporter molecule in response to protein activity changes may be detected
and isolated. In the case of engineering a reporter molecule binding site
that is reactive, a reactive group may be engineered into the site (such
as a thiol) and ability to covalently bind the reporter molecule may be
assayed. A biosensor can then be produced by combining the reporter
molecule with the optimized selectivity component containing the
engineered site.

[0128] In other embodiments, a library of mutants is generated from a
degenerate oligonucleotide sequence. There are many ways by which the
library may be generated from a degenerate oligonucleotide sequence.
Chemical synthesis of a degenerate gene sequence may be carried out in an
automatic DNA synthesizer, and the synthetic genes may then be ligated
into an appropriate vector for expression. One purpose of a degenerate
set of genes is to provide, in one mixture, all of the sequences encoding
the desired set of potential protein sequences. The synthesis of
degenerate oligonucleotides is well known in the art (see for example,
Narang, S A (1983) Tetrahedron 39:3; Itakura et al., (1981) Recombinant
DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton,
Amsterdam: Elsevier pp. 273-289; Itakura et al., (1984) Annu. Rev.
Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike et al.,
(1983) Nucleic Acid Res. 11:477). Such techniques have been employed in
the directed evolution of other proteins (see, for example, Scott et al.,
(1990) Science 249:386-390; Roberts et al., (1992) Proc. Natl. Acad. Sci.
USA 89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et
al., (1990) Proc. Natl. Acad. Sci. USA 87: 6378-6382; as well as U.S.
Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

[0131] In still other embodiments, the selectivity component may be an
aptamer.

[0132] Aptamers are oligonucleotides that are selected to bind
specifically to a desired molecular structure. Aptamers typically are the
products of an affinity selection process similar to the affinity
selection of phage display (also known as in vitro molecular evolution).
The process involves performing several tandem iterations of affinity
separation, e.g., using a solid support to which the desired immunogen is
bound, followed by polymerase chain reaction (PCR) to amplify nucleic
acids that bound to the immunogens. Each round of affinity separation
thus enriches the nucleic acid population for molecules that successfully
bind the desired immunogen. In this manner, a random pool of nucleic
acids may be "educated" to yield aptamers that specifically bind target
molecules. Aptamers typically are RNA, but may be DNA or analogs or
derivatives thereof, such as, without limitation, peptide nucleic acids
and phosphorothioate nucleic acids.

[0133] In exemplary embodiments, nucleic acid ligands, or aptamers, may be
prepared using the "SELEX" methodology which involves selection of
nucleic acid ligands which interact with a target in a desirable manner
combined with amplification of those selected nucleic acids. The SELEX
process, is described in U.S. Pat. Nos. 5,475,096 and 5,270,163 and PCT
Application No. WO 91/19813. These references, each specifically
incorporated herein by reference, are collectively called the SELEX
Patents.

[0134] The SELEX process provides a class of products which are nucleic
acid molecules, each having a unique sequence, and each of which has the
property of binding specifically to a desired target compound or
molecule. In various embodiments, target molecules may be, for example,
proteins, carbohydrates, peptidoglycans or small molecules. SELEX
methodology can also be used to target biological structures, such as
cell surfaces or viruses, through specific interaction with a molecule
that is an integral part of that biological structure.

[0135] In its most basic form, the SELEX process may be defined by the
following series of steps:

[0136] 1) A candidate mixture of nucleic acids of differing sequence, is
prepared. The candidate mixture generally includes regions of fixed
sequences (i.e., each of the members of the candidate mixture contains
the same sequences in the same location) and regions of randomized
sequences. The fixed sequence regions are selected either, (a) to assist
in the amplification steps described below, (b) to mimic a sequence known
to bind to the target, or (c) to enhance the concentration of a given
structural arrangement of the nucleic acids in the candidate mixture. The
randomized sequences can be totally randomized (i.e., the probability of
finding a base at any position being one. in four) or only partially
randomized (e.g., the probability of finding a base at any location can
be selected at any level between 0 and 100 percent).

[0137] 2) The candidate mixture is contacted with the selected target
under conditions favorable for binding between the target and members of
the candidate mixture. Under these circumstances, the interaction between
the target and the nucleic acids of the candidate mixture can be
considered as forming nucleic acid-target pairs between the target and
those nucleic acids having the strongest affinity for the target.

[0138] 3) The nucleic acids with the highest affinity for the target are
partitioned from those nucleic acids with lesser affinity to the target.
Because only an extremely small number of sequences (and possibly only
one molecule of nucleic acid) corresponding to the highest affinity
nucleic acids exist in the candidate mixture, it is generally desirable
to set the partitioning criteria so that. a significant amount of the
nucleic acids in the candidate mixture (approximately 5-50%) are retained
during partitioning.

[0139] 4) Those nucleic acids selected during partitioning as having the
relatively higher affinity for the target are then amplified to create a
new candidate mixture that is enriched in nucleic acids having a
relatively higher affinity for the target.

[0140] 5) By repeating the partitioning and amplifying steps above, the
newly formed candidate mixture contains fewer and fewer unique sequences,
and the average degree of affinity of the nucleic-acids to the target
will generally increase. The SELEX-process ultimately may yield a
candidate mixture containing one or a small number of unique nucleic
acids representing those nucleic acids from the original candidate
mixture having the highest affinity to the target molecule.

[0141] The basic SELEX method has been modified to achieve a number of
specific objectives. For example, U.S. Pat. No. 5,707,796 describes the
use of the SELEX process in conjunction with gel electrophoresis to
select nucleic acid molecules with specific structural characteristics,
such as bent DNA. U.S. Pat. No. 5,580,737 describes a method for
identifying highly specific nucleic acid ligands able to discriminate
between closely related molecules, termed CounterSELEX. U.S. Pat. No.
5,567,588 describes a SELEX-based method which achieves highly efficient
partitioning between oligonucleotides having high and low affinity for a
target molecule. U.S. Pat. Nos. 5,496,938 and 5,683,867 describe methods
for obtaining improved nucleic acid ligands after SELEX has been
performed.

[0142] In certain-embodiments, nucleic acid ligands as described herein
may comprise modifications that increase their stability, including, for
example, modifications that provide increased resistance to degradation
by enzymes such as endonucleases and exonucleases, and/or modifications
that enhance or mediate the delivery of the nucleic acid ligand (see,
e.g., U.S. Pat. Nos. 5,660,985 and 5,637,459). Examples of such
modifications include chemical substitutions at the ribose and/or
phosphate and/or base positions. In various embodiments, modifications of
the nucleic acid ligands may include, but are not limited to, those which
provide other chemical groups that incorporate additional charge,
polarizability, hydrophobicity, hydrogen bonding, electrostatic
interaction, and fluxionality to the nucleic acid ligand bases or to the
nucleic acid ligand as a whole. Such modifications include, but are not
limited to, 2'-position sugar modifications, 5-position pyrimidine
modifications, 8-position purine modifications, modifications at
exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo
or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl
phosphate modifications, methylations, unusual base-pairing combinations
such as the isobases isocytidine and isoguanidine and the like.
Modifications may also include 3' and 5' modifications such as capping.
In exemplary embodiments, the nucleic acid ligands are RNA molecules that
are 2'-fluoro (2'-F) modified on the sugar moiety of pyrimidine residues.

[0143] 3.C. Other Exemplary Selectivity Components

[0144] In other embodiments, the selectivity components may be template
imprinted material. Template imprinted materials are structures which
have an outer sugar layer and an underlying plasma-deposited layer. The
outer sugar layer contains indentations or imprints which are
complementary in shape to a desired target molecule or template so as to
allow specific interaction between the template imprinted structure and
the target molecule to which it is complementary. Template imprinting can
be utilized on the surface of a variety of structures, including, for
example, medical prostheses (such as artificial heart valves, artificial
limb joints, contact lenses and stents), microchips (preferably
silicon-based microchips) and components of diagnostic equipment designed
to detect specific microorganisms, such as viruses or bacteria.
Template-imprinted materials are discussed in U.S. Pat. No. 6,131,580,
which is hereby incorporated by reference in its entirety.

[0145] 3.D. Modification of Selectivity Components for Incorporation into
Biosensors and Exemplary Embodiments Wherein the Selectivity Component is
Produced Independently of the Cell or Tissue to be Analyzed

[0146] In certain embodiments, a selectivity component of the invention
may contain a chemical handle which facilitates its isolation,
immobilization, identification, or detection and/or which increases its
solubility. In various embodiments, chemical handles may be a
polypeptide, a polynucleotide, a carbohydrate, a polymer, or a chemical
moiety and combinations or variants thereof. In certain embodiments,
exemplary chemical handles, include, for example, glutathione
S-transferase (GST); protein A, protein G, calmodulin-binding peptide,
thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His,
poly His-Asp or FLAG tags. Additional exemplary chemical handles include
polypeptides that alter protein localization in vivo, such as signal
peptides, type III secretion system-targeting peptides, transcytosis
domains, nuclear localization signals, etc. In various embodiments, a
selectivity component of the invention may comprise one or more chemical
handles, including multiple copies of the same chemical handle or two or
more different chemical handles. It is also within the scope of the
invention to include a linker (such as a polypeptide sequence or a
chemical moiety) between a selectivity component of the invention and the
chemical handle in order to facilitate construction of the molecule or to
optimize its structural constraints.

[0147] In another embodiment, a selectivity component of the invention may
be modified so that its rate of traversing the cellular membrane is
increased. For example, the selectivity component may be attached to a
peptide which promotes "transcytosis," e.g., uptake of a polypeptide by
cells. The peptide may be a portion of the HIV transactivator (TAT)
protein, such as the fragment corresponding to residues 37-62 or 48-60 of
TAT, portions which have been observed to be rapidly taken up by a cell
in vitro (Green and Loewenstein, (1989) Cell 55:1179-1188).
Alternatively, the internalizing peptide may be derived from the
Drosophila antennapedia protein, or homologs thereof. The 60 amino acid
long homeodomain of the homeo-protein antennapedia has been demonstrated
to translocate through biological membranes and can facilitate the
translocation of heterologous polypeptides to which it-is coupled. Thus,
selectivity components may be fused to a peptide consisting of about
amino acids 42-58 of Drosophila antennapedia or shorter fragments for
transcytosis (Derossi et al. (1996) J Biol Chem 271:18188-18193; Derossi
et al. (1994) J Biol Chem 269:10444-10450; and Perez et al. (1992) J Cell
Sci 1.02:717-722). The transcytosis polypeptide may also be a
non-naturally-occurring membrane-translocating sequence (MTS), such as
the peptide sequences disclosed in U.S. Pat. No. 6,248,558.

[0148] In still other embodiments, the selectivity component may comprise
a fusion protein of any of the above-described polypeptide selectivity
components containing at least one domain which increases its solubility
and/or facilitates its purification, identification, detection, targeting
and/or delivery. Exemplary domains, include, for example, glutathione
S-transferase (GST), protein A, protein G, calmodulin-binding peptide,
thioredoxin, maltose binding protein, HA, myc, poly arginine, poly His,
poly His-Asp or FLAG fusion proteins and tags. Additional exemplary
domains include domains that alter protein localization in vivo, such as
signal peptides, type III secretion system-targeting peptides,
transcytosis domains, nuclear localization signals, and targeting
moieties, i.e. proteins specific for a target molecule, etc. In various
embodiments, a polypeptide of the invention may comprise one or more
heterologous fusions. Polypeptides may contain multiple copies of the
same fusion domain or may contain fusions to two or more different
domains. The fusions may occur at the N-terminus of the polypeptide, at
the C-terminus of the polypeptide, or at both the N- and C-terminus of
the polypeptide. Linker sequences between a polypeptide of the invention
and the fusion domain may be included in order to facilitate construction
of the fusion protein or to optimize protein expression or structural
constraints of the fusion protein.

[0149] In exemplary embodiments, the dissociation constant of the
selectivity component for a target molecule is optimized to allow real
time monitoring of the presence and/or concentration of the analyte in a
given patient, sample, or environment.

[0150] The selectivity components (for example, phage, antibodies,
antibody fragments, aptamers, etc.) may be affixed to a suitable
substrate by a number of known methods. Typically the surface of the
substrate is functionalized in some manner, so that a crosslinking
compound or compounds may covalently link the selectivity component to
the substrate. For example, a substrate functionalized with carboxyl
groups may be linked to free amines in the selectivity components using
EDC or by other common chemistries, such as by linking with
N-hydroxysuccinimide. A variety of crosslinking chemistries are
commercially available, for instance, from Pierce of Rockford, Ill.

[0151] For attachment of the sensor units to surfaces there are a number
of traditional attachment technologies. For example, activated carboxyl
groups on the substrate will link the sensor units to the substrate via
--NH2 groups on the selectivity component of the biosensor. The substrate
of the array may be either organic or inorganic, biological or
non-biological, or any combination of these materials. Numerous materials
are suitable for use as a substrate for the sensor units of the
invention. For instance, the substrate of the invention sensors can
comprise a material selected from a group consisting of silicon, silica,
quartz, glass, controlled pore glass, carbon, alumina, titania, tantalum
oxide, germanium, silicon nitride, zeolites, and gallium arsenide. Many
metals such as gold, platinum, aluminum, copper, titanium, and their
alloys are also options for substrates of the array. In addition, many
ceramics and polymers may also be used as substrates. Polymers which may
be used as substrates include, but are not limited to, the following:
polystyrene; poly(tetra)fluoroethylene (PTFE); polyvinylidenedifluoride;
polycarbonate; polymethylmethacrylate; polyvinylethylene;
polyethyleneimine; poly(etherether)ketone; polyoxymethylene (POM);
polyvinylphenol; polylactides; polymethacrylimide (PMI);
polyalkenesulfone (PAS); polypropylethylene, polyethylene;
polyhydroxyethylmethacrylate (HEMA); polydimethylsiloxane;
polyacrylamide; polyimide; and block copolymers. Preferred substrates for
the array include silicon, silica, glass, and polymers. The substrate on
which the sensors reside may also be a combination of any of the
aforementioned substrate materials.

[0152] A biosensor of the present invention may optionally further
comprise a coating between the substrate and the bound biosensor
molecule. This coating may either be formed on the substrate or applied
to the substrate. The substrate can be modified with a coating by using
thin-film technology based, for instance, on physical vapor deposition
(PVD), plasma-enhanced chemical vapor deposition (PECVD), or thermal
processing. Alternatively, plasma exposure can be used to directly
activate or alter the substrate and create a coating. For instance,
plasma etch procedures can be used to oxidize a polymeric surface (for
example, polystyrene or polyethylene to expose polar functionalities such
as hydroxyls, carboxylic acids, aldehydes and the like) which then acts
as a coating.

[0154] The substrate surface shall comprise molecules of formula
X(a)-R(b)-Y(c), wherein R is a spacer, X is a functional group that binds
R to the surface, Y is a functional group for binding to the biosensor,
(a) is an integer from 0 to about 4, (b) is either 0 or 1, and (c) is an
integer not equal to 0. Note that when both (a) and (b) are zero, the
substrate surface comprises functional groups Y as would be seen, for
example, with polymeric substrates or coatings. When (a) and (b) are not
equal to 0, then X(a)-R(b)-Y(c) describes, for example, monolayers such
as a self assembled monolayers that form on a metal surface.
X(a)-R(b)-Y(c) may also describe such compounds as
3-aminopropyltrimethoxysilane, wherein X is --Si(OMe)3, R is
--CH2CH2CH2--, and Y is --NH2. This compound is known
to coat porous glass surfaces to form an aminopropyl derivative of the
glass. Biochem. Biophys. Act., 1970, 212, 1; J. Chromatography, 1974, 97,
39.

[0155] Other definitions for F, X, and Y include the following. R
optionally comprises a linear or branched hydrocarbon chain from about 1
to about 400 carbons long. The hydrocarbon chain may comprise an alkyl,
aryl, alkenyl, alkynyl, cycloalkyl, alkaryl, aralkyl group, or any
combination thereof. If (a) and (c) are both equal to one, then R is
typically an alkyl chain from about 3 to about 30 carbons long. In a
preferred embodiment, if (a) and (b) are both equal to one, then R is an
alkyl, chain from about 8 to about 22 carbons long and is, optionally, a
straight alkane. However, it is also contemplated that in an alternative
embodiment, R may readily comprise a linear or branched hydrocarbon chain
from about 2 to about 400 carbons long and be interrupted by at least one
hetero atom. The interrupting hetero groups can include --O--,
--CONH2, --CONHCO--, --NH--, --CSNH--, --CO--, --CS--, --S--,
--SO--, --(OCH2CH2)n- (where n=1-20), --(CF2)n (where
n=1-22), and the like. Alternatively, one or more of the hydrogen
moieties of R can be substituted with deuterium. In alternative
embodiments, R may be more than about 400 carbons long.

[0156] X may be chosen as any group which affords chemisorption or
physisorption of the monolayer onto the surface of the substrate (or the
coating, if present). When the substrate or coating is a-metal or metal
alloy, X, at least prior to incorporation into the monolayer, can in one
embodiment be chosen to be an asymmetrical or symmetrical disulfide,
sulfide, diselenide, selenide, thiol, isonitrile, selenol, a trivalent
phosphorus compound, isothiocyanate, isocyanate, xanthanate,
thiocarbamate, a phosphine, an amine, thio acid or a dithio acid. This
embodiment is especially preferred when a coating or substrate is used
that is a noble metal such as gold, silver, or platinum.

[0157] If the substrate is a material such as silicon, silicon oxide,
indium tin oxide, magnesium oxide, alumina, quartz, glass, or silica,
then, in one embodiment, the biosensor may comprise an X that, prior to
incorporation into said monolayer, is a monohalosilane, dihalosilane,
tihalosilane, trialkoxysilane, dialkoxysilane, or a monoalkoxysilane.
Among these silanes, trichlorosilane and trialkoxysilane are exemplary.

[0158] In certain embodiments, the substrate is selected from the group
consisting of silicon, silicon dioxide, indium tin oxide, alumina, glass,
and titania; and X is selected from the group consisting of a
monohalosilane, dihalosilane, tihalosilane, trichlorosilane,
trialkoxysilane, dialkoxysilane, monoalkoxysilane, carboxylic acids, and
phosphates.

[0159] In another embodiment, the substrate of the sensor is silicon and X
is an olefin.

[0160] In still another embodiment, the coating (or the substrate if no
coating is present) is titania or tantalum oxide and X is a phosphate.

[0161] In other embodiments, the surface of the substrate (or coating
thereon) is composed of a material such as titanium oxide; tantalum
oxide, indium tin oxide, magnesium oxido, or alumina where X is a
carboxylic acid or alkylphosphoric acid. Alternatively, if the surface of
the substrate (or coating thereon) of the sensor is copper, then X may
optionally. be a hydroxamic acid.

[0162] If the substrate used in the invention is a polymer, then in many
cases a coating on the substrate such as a copper coating will be
included in the sensor. An appropriate functional group X for the coating
would then be chosen for use in the sensor. In an alternative embodiment
comprising a polymer substrate, the surface of the polymer may be plasma
modified to expose desirable surface functionalities for monolayer
formation. For instance, EP 780423 describes the use of a monolayer
molecule that has an alkene X functionality on a plasma exposed surface.
Still another possibility for the invention sensor comprised of a polymer
is that the surface of the polymer on which the monolayer is formed is
functionalized by copolymerization of appropriately functionalized
precursor molecules.

[0163] Another possibility is that prior to incorporation into the
monolayer, X can be a free radical-producing moiety. This functional
group is especially appropriate when the surface on which the monolayer
is formed is a hydrogenated silicon surface. Possible free-radical
producing moieties include, but are not limited to, diacylperoxides,
peroxides, and azo compounds. Alternatively, unsaturated moieties such as
unsubstituted alkenes, alkynes, cyanocompounds and isonitrile compounds
can be used for-X, if the reaction with X is accompanied by ultraviolet,
infrared, visible, or microwave radiation.

[0164] In alternative embodiments, X may be a hydroxyl, carboxyl, vinyl,
sulfonyl, phosphoryl, silicon hydride, or an amino group.

[0165] The component Y is a functional group responsible for binding a dye
containing sensor onto the substrate. In one embodiment, the Y group is
either highly reactive (activated) towards the dye containing sensor or
is easily converted into such an activated form. In certain embodiments,
the coupling of Y with the selectivity component of the biosensor occurs
readily under normal physiological conditions. The functional group Y may
either form a covalent linkage or a noncovalent linkage with the
selectivity component of the biosensor. In other embodiments, the
functional group Y forms a covalent linkage with the selectivity
component of the biosensor. It is understood that following the
attachment of the selectivity component of the biosensor to Y, the
chemical nature of Y may have changed. Upon attachment of the biosensor,
Y may even have been removed from the organic linker.

[0166] In one embodiment of the sensor of the present invention, Y is a
functional group that is activated in situ. Possibilities for this type
of functional group include, but are not limited to, such simple moieties
such as a hydroxyl, carboxyl, amino, aldehyde, carbonyl, methyl,
methylene, alkene, allyne, carbonate, aryliodide, or a vinyl group.
Appropriate modes of activation would be obvious to one skilled in the
art. Alternatively, Y can comprise a functional group that requires
photoactivation prior to becoming activated enough to trap the protein
capture agent.

[0168] In an alternative embodiment, the functional group Y is selected
from the group of simple functional moieties. Possible Y functional
groups include, but are not limited to --OH, --NH2, --COOH, --COOR,
--RSR, --PO4-3, --OSO3-2, --SO3.sup.-,
--COONR2, SOO.sup.-, --CN, --NR2, and the like.

[0169] In another embodiment, one or more biosensor species may be bound
to discrete beads or microspheres. The microspheres typically are either
carboxylated or avidin-modified so that proteins, such as antibodies,
non-antibody receptors and variants and fragments thereof, may be readily
attached to the beads by standard chemistries. In an exemplary
embodiment, the selectivity components are scFv fragments. The scFv
fragments may be bound to carboxylated beads by one of many linking
chemistries, such as, for example, EDC chemistry, or bound to
avidin-coated beads by first biotinylating the scFv fragment by one of
many common biotinylation chemistries, such as, for example, by
conjugation with sulfo-NHS-LC-biotin.

[0170] In another embodiment, two or more biosensors are affixed to one or
more supports at discrete locations (that is, biosensors having a first
specificity are affixed at a first spatial location, biosensors having a
second specificity are affixed at a second spatial location, etc.). In
one embodiment, the biosensors are affixed to a substrate in a tiled
array, with each biosensor represented in one or more positions in the
tiled array. The spatial configuration of the substrate or substrates may
be varied so long as each biosensor species is bound at detectably
discrete locations. The substrate and tiled biosensor pattern typically
is planar, but may be any geometric configuration desired. For instance,
the substrate may be a strip or cylindrical, as illustrated in U.S. Pat.
No. 6,057,100. In exemplary embodiments, the substrate may be glass- or
other silicic compositions; such as those used in the semiconductor
industry.

[0171] Fabrication of the substrate may be by one of many well-known
processes. In various embodiments, the biosensors of the array may be
associated with the same reporter molecule or may be associated with
different reporter molecules. Identification of a biosensor that
interacts with a target molecule may be based on the signal from the
reporter molecule, the location of the biosensor on the array, or a
combination thereof. Arrays may be used in association with both the in
vitro and in vivo applications of the invention.

[0172] In various embodiments, the arrays may comprise any of the
biosensors described herein, including, for example, arrays of biosensors
wherein the selectivity components are polypeptides (including,
antibodies and variants or fragments thereof), polynucleotides (i.e.,
aptamers), template imprinted materials, organic binding elements, and
inorganic binding elements. The arrays may comprise one type of biosensor
or a mixture of different types of biosensors (e.g., a mixture of
biosensors having polypeptide and polynucleotide selectivity components).
Protein microarrays are described, for example, in PCT Publication WO
00/04389, incorporated herein by reference. Examples of commercially
available protein microarrays are those of Zyomyx of Hayward, Calif.,
Ciphergen Biosystems, Inc. of Fremont, Calif. and Nanogen, Inc. of San
Diego, Calif. Nucleic acid microarrays are described, for example, in
U.S. Pat. Nos. 6,261,776 and 5,837,832. Examples of commercially
available nucleic acid microarrays are those of Affymetrix, Inc. of Santa
Clara, Calif., BD Biosciences Clontech of Palo Alto, Calif. and
Sigma-Aldrich Corp. of St. Louis, Mo.

[0173] 3.E. Exemplary Embodiments Wherein the Selectivity Component is
Expressed in the Cell or Tissue to be Analyzed

[0174] In other embodiments, the selectivity component is expressed within
the cell or organism or subject to be analyzed. The expression methods
described below may also be used to express a selectivity component in a
host cell that is then isolated and purified for use in the methods,
wherein the biosensor is generated from a source external to the cell or
tissue to be analyzed.

[0175] Generally, a nucleic acid encoding a selectivity component is
introduced into a host cell, such as by transfection or infection, and
the host cell is cultured under conditions allowing expression of the
selectivity component. Methods of introducing nucleic acids into
prokaryotic and eukaryotic cells are well known in the art. Suitable
media for mammalian and prokaryotic host cell culture are well known in
the art. In some instances, the nucleic acid encoding the subject
polypeptide is under the control of an inducible promoter, which is
induced once the host cells comprising the nucleic acid have divided a
certain number of times. For example, where a nucleic acid is under the
control of a beta-galactose operator and repressor, isopropyl
beta-D-thiogalactopyranoside (IPTG) is added to the culture when the
bacterial host cells have attained a density of about OD600
0.45-0.60. The culture is then grown for some more time to give the host
cell the time to synthesize the polypeptide. Cultures are then typically
frozen and may be stored frozen for some time, prior to isolation and
purification of the polypeptide.

[0176] Thus, a nucleotide sequence encoding all or part of a selectivity
component may be used to produce a recombinant form of a selectivity
component via microbial or eukaryotic cellular processes. Ligating the
sequence into a polynucleotide construct, such as an expression vector,
and transforming, infecting, or transfecting into hosts, either
eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial
cells), are standard procedures. Similar procedures, or modifications
thereof, may be employed to prepare recombinant polypeptides by microbial
means or tissue-culture technology in accord with the subject invention.

[0177] Other embodiments of nucleic acid sequences encoding the
selectivity components, as well as vectors, host cells, and cultures
thereof are further described below.

[0179] In another embodiment, a signal peptide sequence is added to the
construct, such that the selectivity component is secreted from cells.
Such signal peptides are well known in the art.

[0180] In one embodiment, the powerful phage T5 promoter, that is
recognized by E. coli RNA polymerase is used together with a lac operator
repression module to provide tightly regulated, high level expression or
recombinant proteins in E. coli. In this system, protein expression is
blocked in the presence of high levels of lac repressor.

[0181] In one embodiment, the DNA is operably linked to a first promoter
and the bacterium further comprises a second DNA encoding a first
polymerase which is capable of mediating transcription from the first
promoter, wherein the DNA encoding the first polymerase is operably
linked to a second promoter. In a preferred embodiment, the second
promoter is a bacterial promoter, such as those delineated above. In an
even more preferred embodiment, the polymerase is a bacteriophage
polymerase, e.g., SP6, T3, or T7 polymerase and the first promoter is a
bacteriophage promoter, e.g., an SP6, T3, or T7 promoter, respectively.
Plasmids comprising bacteriophage promoters and plasmids encoding
bacteriophage polymerases can be obtained commercially, e.g., from
Promega Corp. (Madison, Wis.) and InVitrogen (San Diego, Calif.), or can
be obtained directly from the bacteriophage using standard recombinant
DNA techniques (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning:
A Laboratory Manual, Cold Spring Laboratory Press, 1989). Bacteriophage
polymerases and promoters are further described, e.g., in the following
references: Sagawa, H. et al. (1996) Gene 168:37; Cheng, X. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:4034; Dubendorff, J. W. and F. W. Studier
(1991) Journal of Molecular Biology 219:45; Bujarski, J. J. and P.
Kaesberg (1987) Nucleic Acids Research 15:1337; and Studier, F. W. et al.
(1990) Methods in Enzymology 185:60). Such plasmids can further be
modified according to the specific embodiment of the invention.

[0182] In another embodiment, the bacterium further comprises a DNA
encoding a second polymerase which is capable of mediating transcription
from the second promoter, wherein the DNA encoding the second polymerase
is operably linked to a third promoter. In a preferred embodiment, the
third promoter is a bacterial promoter. However, more than two different
polymerases and promoters could be introduced in a bacterium to obtain
high levels of transcription. The use of one or more polymerase for
mediating transcription in the bacterium can provide a significant
increase in the amount of polypeptide in the bacterium relative to a
bacterium in which the DNA is directly under the control of a bacterial
promoter. The selection of the system to adopt will vary depending on the
specific use of the invention, e.g., on the amount of protein that one
desires to produce.

[0183] When using a prokaryotic host cell, the host cell may include a
plasmid which expresses an internal T7 lysozyme, e.g., expressed from
plasmid. Lysis of such host cells liberates the lysozyme which then
degrades the bacterial membrane.

[0184] Other sequences that may be included in a vector for expression in
bacterial or other prokaryotic cells include a synthetic ribosomal
binding site; strong transcriptional terminators, e.g., t0 from phage
lambda and t4 from the rrnB operon in E. coli, to prevent read through
transcription and ensure stability of the expressed polypeptide; an
origin of replication, e.g., ColE1; and beta-lactamase gene, conferring
ampicillin resistance.

[0186] A number of vectors exist for the expression of recombinant
proteins in yeast. For instance, YEP24, YIPS, YEP51, YEP52, pYES2, and
YRP17 are cloning and expression vehicles useful in the introduction of
genetic constructs into S. cerevisiae (see, for example, Broach et al.,
(1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye
Academic Press, p. 83). These vectors may replicate in E. coli due the
presence of the pBR322 ori, and in S. cerevisiae due to the replication
determinant of the yeast 2 micron plasmid. In addition, drug resistance
markers such as ampicillin may be used.

[0187] In certain embodiments, mammalian expression vectors contain both
prokaryotic sequences to facilitate the propagation of the vector in
bacteria, and one or more eukaryotic transcription units that are
expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV,
pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg
derived vectors are examples of mammalian expression vectors suitable for
transfection of eukaryotic cells. Some of these vectors are modified with
sequences from bacterial plasmids, such as pBR322, to facilitate
replication and drug resistance selection in both prokaryotic and
eukaryotic cells. Alternatively, derivatives of viruses such as the
bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo,
pREP-derived and p205) can be used for transient expression of proteins
in eukaryotic cells. The various methods employed in the preparation of
the plasmids and transformation of host organisms are well known in the
art. For other suitable expression systems for both prokaryotic and
eukaryotic cells, as well as general recombinant procedures, see
Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch
and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and
17. In some instances, it may be desirable to express the recombinant
protein by the use of a baculovirus expression system. Examples of such
baculovirus expression systems include pVL-derived vectors (such as
pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and
pFastBac-derived vectors.

[0188] In another variation, protein production may be achieved using in
vitro translation systems. In vitro translation systems are, generally, a
translation system which is a cell-free extract comprising at least the
minimum elements necessary for translation of an RNA molecule into a
protein. An in vitro translation system typically comprises at least
ribosomes, tRNAs, initiator methionyl-tRNAMet, proteins or complexes
involved in translation, e.g., eIF2, eIF3, the cap-binding (CB) complex,
comprising the cap-binding protein (CBP) and eukaryotic initiation factor
4F (eIF4F). A variety of in vitro translation systems are well known in
the art and include commercially available kits. Examples of in vitro
translation systems include eukaryotic lysates, such as rabbit
reticulocyte lysates, rabbit oocyte lysates, human cell lysates, insect
cell lysates and wheat germ extracts. Lysates are commercially available
from manufacturers such as Promega Corp., Madison, Wis.; Stratagene, La
Jolla, Calif.; Amersham, Arlington Heights, Ill.; and GIBCO/BRL, Grand
Island, N.Y. In vitro translation systems typically comprise
macromolecules, such as enzymes, translation, initiation and elongation
factors, chemical reagents, and ribosomes. In addition, an in vitro
transcription system may be used. Such systems typically comprise at
least an RNA polymerase holoenzyme, ribonucleotides and any necessary
transcription initiation, elongation and termination factors. An RNA
nucleotide for in vitro translation may be produced using methods known
in the art. In vitro transcription and translation may be coupled in a
one-pot reaction to produce proteins from one or more isolated DNAs.

[0189] When expression of a carboxy terminal fragment of a polypeptide is
desired, i.e. a truncation mutant, it may be necessary to add a start
codon (ATG) to the oligonucleotide fragment comprising the desired
sequence to be expressed. It is well known in the art that a methionine
at the N-terminal position may be enzymatically cleaved by the use of the
enzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli
(Ben-Bassat et al., (1987) J. Bacteriol. 169:751-757) and Salmonella
typhimurium and its in vitro activity has been demonstrated on
recombinant proteins (Miller et al., (1987) Proc. Natl. Acad. Sci. USA
84:2718-1722). Therefore, removal of an N-terminal methionine, if
desired, may be achieved either in vivo by expressing such recombinant
polypeptides in a host which produces MAP (e.g., E. coli or CM89 or S.
cerevisiae), or in vitro by use of purified MAP (e.g., procedure of
Miller et al.).

[0191] An alternative expression system which can be used to express a
polypeptide is an insect system. In one such system, Autographa
californica nuclear polyhedrosis virus (AcNPV) is used as a vector to
express foreign genes. The virus grows in Spodoptera frugiperda cells.
The PGHS-2 sequence may be cloned into non-essential regions (for example
the polyhedrin gene) of the virus and placed under control of an AcNPV
promoter (for example the polyhedrin promoter). Successful insertion of
the coding sequence will result in inactivation of the polyhedrin gene
and production of non-occluded recombinant virus (i.e., virus lacking the
proteinaceous coat coded for by the polyhedrin gene). These recombinant
viruses are then used to infect Spodoptera frugiperda cells in which the
inserted gene is expressed. (e.g., see Smith et al., 1983, J. Virol.,
46:584, Smith, U.S. Pat. No. 4,215,051).

[0192] In a specific embodiment of an insect system, the DNA encoding the
subject polypeptide is cloned into the pBlueBacIII recombinant transfer
vector (Invitrogen, San Diego, Calif.) downstream of the polyhedrin
promoter and transfected into Sf9 insect cells (derived from Spodoptera
frugiperda ovarian cells, available from Invitrogen, San Diego, Calif.)
to generate recombinant virus. After plaque purification of the
recombinant virus high-titer viral stocks are prepared that in turn would
be used to infect Sf9 or High Five® (BTI-TN-5B1-4 cells derived from
Trichoplusia ni egg cell homogenates; available from Invitrogen, San
Diego, Calif.) insect cells, to produce large quantities of appropriately
post-translationally modified subject polypeptide. Although it is
possible that these cells themselves could be directly useful for drug
assays, the subject polypeptides prepared by this method can be used for
in vitro assays.

[0193] In another embodiment, the subject polypeptides are prepared in
transgenic animals, such that in certain embodiments, the polypeptide is
secreted, e.g., in the milk of a female animal.

Viral vectors may also be used for efficient in vitro introduction of a
nucleic acid into a cell. Infection of cells with a viral vector has the
advantage that a large proportion of the targeted cells can receive the
nucleic acid. Additionally, polypeptides encoded by genetic material in
the viral vector, e.g., by a nucleic acid contained in the viral vector,
are expressed efficiently in cells that have taken up viral vector
nucleic acid.

[0194] Retrovirus vectors and adeno-associated virus vectors are generally
understood to be the recombinant gene delivery system of choice for the
transfer of exogenous genes in vivo, particularly into mammals. These
vectors provide efficient delivery of genes into cells, and the
transferred nucleic acids are stably integrated into the chromosomal DNA
of the host. A major prerequisite for the use of retroviruses is to
ensure the safety of their use, particularly with regard to the
possibility of the spread of wild-type virus in the cell population. The
development of specialized cell lines (termed "packaging cells") which
produce only replication-defective retroviruses has increased the utility
of retroviruses for gene therapy, and defective retroviruses are well
characterized for use in gene transfer for gene therapy purposes (see
Miller, A. D. (1990) Blood 76:271). Thus, recombinant retrovirus can be
constructed in which part of the retroviral coding sequence (gag, pol,
env) has been replaced by nucleic acid encoding one of the antisense E6AP
constructs, rendering the retrovirus replication defective. The
replication defective retrovirus is then packaged into virions which can
be used to infect a target cell through the use of a helper virus by
standard techniques. Protocols for producing recombinant retroviruses and
for infecting cells in vitro or in vivo with such viruses can be found in
Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.)
Greene Publishing Associates, (1989), Sections 9.10-9.14, and other
standard laboratory manuals. Examples of suitable retroviruses include
pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
Examples of suitable packaging virus lines for preparing both ecotropic
and amphotropic retroviral systems include Crip, Cre and Am. Retroviruses
have been used to introduce a variety of genes into many different cell
types, including neural cells, epithelial cells, endothelial cells,
lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or
in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398;
Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson
et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al.
(1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc.
Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad.
Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van
Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et
al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.
Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.
150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT
Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO
89/05345; and PCT Application WO 92/07573).

[0195] In choosing retroviral vectors as a gene delivery system for
nucleic acids encoding the subject polypeptides, it is important to note
that a prerequisite for the successful infection of target cells by most
retroviruses, and therefore of stable introduction of the genetic
material, is that the target cells must be dividing. In general, this
requirement will not be a hindrance to use of retroviral vectors. In
fact, such limitation on infection can be beneficial in circumstances
wherein the tissue (e.g., nontransformed cells) surrounding the target
cells does not undergo extensive cell division and is therefore
refractory to infection with retroviral vectors.

[0196] Furthermore, it has been shown that it is possible to limit the
infection spectrum of retroviruses and consequently of retroviral-based
vectors, by modifying the viral packaging proteins on the surface of the
viral particle (see, for example, PCT publications WO93/25234,
WO94/06920, and WO94/11524). For instance, strategies for the
modification of the infection spectrum of retroviral vectors include:
coupling antibodies specific for cell surface antigens to the viral env
protein (Roux et al. (1989) Proc. Natl. Acad. Sci. USA 86:9079-9083;
Julan et al. (1992) J. Gen Virol 73:3251-3255; and Goud et al. (1983)
Virology 163:251-254); or coupling cell surface ligands to the viral env
proteins (Neda et al. (1991) J Biol Chem 266:14143-14146). Coupling can
be in the form of the chemical cross-linking with a protein or other
variety (e.g., lactose to convert the env protein to an
asialoglycoprotein), as well as by generating chimeric proteins (e.g.,
single-chain antibody/env chimeric proteins). This technique, while
useful to limit or otherwise direct the infection to certain tissue
types, and can also be used to convert an ecotropic vector in to an
amphotropic vector.

[0197] Moreover, use of retroviral gene delivery can be further enhanced
by the use of tissue- or cell-specific transcriptional regulatory
sequences which control expression of the genetic material of the
retroviral vector.

[0198] Another viral gene delivery system utilizes adenovirus-derived
vectors. The genome of an adenovirus can be manipulated such that it
encodes a gene product of interest, but is inactive in terms of its
ability to replicate in a normal lytic viral life cycle (see, for
example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al.
(1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155).
Suitable adenoviral vectors derived from the adenovirus strain Ad type 5
d1324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are well
known to those skilled in the art. Recombinant adenoviruses can be
advantageous in certain circumstances in that they are capable of
infecting non-dividing cells and can be used to infect a wide variety of
cell types, including airway epithelium (Rosenfeld et al. (1992) cited
supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad.
Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl.
Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992)
Proc. Natl. Acad. Sci. USA 89:2581-2584). Furthermore, the virus particle
is relatively stable and amenable to purification and concentration, and,
as above, can be modified so as to affect the spectrum of infectivity.
Additionally, introduced adenoviral DNA (and foreign DNA contained
therein) is not integrated into the genome of a host cell but remains
episomal, thereby avoiding potential problems that can occur as a result
of insertional mutagenesis in situations where introduced DNA becomes
integrated into the host genome (e.g., retroviral DNA). Moreover, the
carrying capacity of the adenoviral genome for foreign DNA is large (up
to 8 kilobases) relative to other gene delivery vectors (Berkner et al.,
supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most
replication-defective adenoviral vectors currently in use and therefore
favored by the present invention are deleted for all or parts of the
viral E1 and E3 genes but retain as much as 80% of the adenoviral genetic
material (see, for example, Jones et al. (1979) Cell 16:683; Berkner et
al., supra; and Graham et al. in Methods in Molecular Biology, E. J.
Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp. 109-127).
Expression of the inserted genetic material can be under control of, for
example, the E1A promoter, the major late promoter (MLP) and associated
leader sequences, the E3 promoter, or exogenously added promoter
sequences.

[0199] Yet another viral vector system useful for delivery of genetic
material encoding the subject polypeptides is the adeno-associated virus
(AAV). Adeno-associated virus is a naturally occurring defective virus
that requires another virus, such as an adenovirus or a herpes virus, as
a helper virus for efficient replication and a productive life cycle.
(see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992)
158:97-129). It is also one of the few viruses that may integrate its DNA
into non-dividing cells, and exhibits a high frequency of stable
integration (see for example Flotte et al. (1992) Am. J. Respir. Cell.
Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and
McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors comprising as
little as 300 base pairs of AAV can be packaged and can integrate. Space
for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that
described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be
used to introduce DNA into cells. A variety of nucleic acids have been
introduced into different cell types using AAV vectors (see for example
Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin
et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol.
Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51:611-619; and
Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

[0200] In particular, a AAV delivery system suitable for targeting muscle
tissue has been developed by Gregorevic, et al., Nat Med. 2004 August;
10(8):828-34. Epub 2004 Jul. 25, which is able to `home-in` on muscle
cells and does not trigger an immune system response. The delivery system
also includes use of a growth factor, VEGF, which appears to increase
penetration into muscles of the gene therapy agent.

[0201] Other viral vector systems may be derived from herpes virus,
vaccinia virus, and several RNA viruses.

[0202] In addition to viral transfer methods, such as those illustrated
above, non-viral methods can also be employed to cause expression of
nucleic acids encoding the subject polypeptides, e.g. in a cell in vitro
or in the tissue of an animal. Most nonviral methods of gene transfer
rely on normal mechanisms used by mammalian cells for the uptake and
intracellular transport of macromolecules. In preferred embodiments,
non-viral gene delivery systems of the present invention rely on
endocytic pathways for the uptake of genetic material by the targeted
cell. Exemplary gene delivery systems of this type include liposomal
derived systems, polylysine conjugates, and artificial viral envelopes.

[0204] In yet another illustrative embodiment, the gene delivery system
comprises an antibody or cell surface ligand which is cross-linked with a
gene binding agent such as polylysine (see, for example, PCT publications
WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180). For
example, genetic material encoding the subject chimeric polypeptides can
be used to transfect hepatocytic cells in vivo using a soluble
polynucleotide carrier comprising an asialoglycoprotein conjugated to a
polycation, e.g., polylysine (see U.S. Pat. No. 5,166,320). It will also
be appreciated that effective delivery of the subject nucleic acid
constructs via mediated endocytosis can be improved using agents which
enhance escape of the gene from the endosomal structures. For instance,
whole adenovirus or fusogenic peptides of the influenza HA gene product
can be used as part of the delivery system to induce efficient disruption
of DNA-comprising endosomes (Mulligan et al. (1993) Science 260-926;
Wagner et al. (1992) Proc. Natl. Acad. Sci. USA 89:7934; and Christiano
et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122).

[0205] Tissue-specific expression of a selectivity component may be
achieved by use of a construct comprising a tissue-specific promoter.

[0206] 4. Reporters

[0207] The reporter may be any molecule which produces a detectable signal
change in response to an alteration in the environment. For example; the
signal change may be an increase or decrease in signal intensity, or a
change in the type of signal produced. In exemplary embodiments, suitable
reporters include molecules which produce optically detectable signals;
including, for example, fluorescent and chemiluminescent molecules. In
certain embodiments, the reporter molecule is a long wavelength
fluorescent molecule which permits detection of the reporter signal
through a tissue sample, especially non-invasive detection of the
reporter in conjunction with in vivo applications.

[0208] Without being bound by theory, in certain embodiments, the reporter
molecule is a pH sensitive fluorescent dye (pH sensor dye) which shows a
spectral change upon interaction of a selectivity component with a target
molecule. Interaction of the selectivity component with a target molecule
may lead to a shift in the pH of the microenvironment surrounding the
selectivity component due to the composition of acidic and basic residues
on the selectivity and/or target molecules. In turn, the shift in the pH
microenvironment leads to a detectable spectral change in the signal of
the pH sensitive fluorescent dye molecule associated with the selectivity
component. In exemplary embodiments, a pH sensitive dye is selected with
an appropriate pKa to lead to an optimal spectral change upon binding of
the particular selectivity component/target molecule combination. A
variety of pH sensitive dyes suitable for use in accordance with the
invention are commercially available. In exemplary embodiments, pH
sensitive dyes include, for example, fluorescein, umbelliferones
(coumarin compounds), pyrenes, resorufin, hydroxy esters, aromatic acids,
styryl dyes, tetramethyl rhodamine dyes, and cyanine dyes, and pH
sensitive derivatives thereof.

[0209] Without being bound by theory, in other embodiments, the reporter
molecule is a polarity sensitive fluorescent dye (polarity sensor dye)
which shows a spectral change upon interaction-of-a-selectivity component
with a target molecule. Interaction of the selectivity component with a
target molecule may lead to a shift in the polarity of the
microenvironment surrounding the selectivity component due to the
composition of polar and/or non-polar residues on the selectivity and/or
target molecules. In turn, the change in the polarity of the
microenvironment leads to a detectable spectral change in the signal of
the polarity sensitive fluorescent dye molecule associated with the
selectivity component. A variety of polarity sensitive dyes suitable for
use in accordance with the invention are commercially available. In
exemplary embodiments, polarity sensitive dyes include, for example,
merocyanine dyes,
5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid
(1,5-IAEDANS), and CPM, and polarity sensitive derivatives of merocyanine
dyes, IAEDANS, and CPM.

[0210] Without being bound by theory, in certain embodiments, the reporter
molecule is a fluorescent dye that is sensitive to changes in the
microviscosity of the local environment (restriction sensor dye).
Interaction of the selectivity component with a target molecule may lead
to a change in the microviscosity in the local environment surrounding
the selectivity component. In turn, the change in microviscosity may lead
to a detectable spectral change in the signal of the mobility sensor dye
molecule associated with the selectivity component. For example, an
increase of microviscosity upon target binding will restrict the dye and
increase the quantum yield of the emitted fluorescence signal. A variety
of restriction sensor dyes suitable for use in accordance with the
invention are commercially available. In exemplary embodiments,
restriction sensor dyes include, for example, monomethine and trimethine
cyanine dyes, and microviscosity sensitive derivatives of monomethine and
trimethine cyanine dyes.

[0211] Without being bound by theory, in certain embodiments, the reporter
molecule is a fluorescent dye that exhibits a spectral change due to a
modification in the tumbling rate of the dye as measured on a nanosecond
time scale (mobility sensor dye). Mobility sensor dye molecules may be
linked to the selectivity component using a linker molecule that permits
free rotation of the dye molecule. Upon interaction of the selectivity
component with a target molecule, the rotation of the dye molecule around
the linker may become restricted leading to a change in the ratio of
parallel to perpendicular polarization of the dye molecule. A change in
polarization of the mobility sensor dye may be detected as a change in
the spectral emission of the dye and can be measured using light
polarization optics for both excitation and emission to determine the
tumbling rate of the dye. Abbott's fluorescence polarization technology
is an exemplary method for determining the polarization of the dye. In
exemplary embodiments, the mobility sensor dye is attached to the
selectivity component using a triple-bond containing linker that extends
the dye away from the surface of the selectivity component. A variety of
mobility sensor dyes suitable for use in accordance with the invention
are commercially available. In exemplary embodiments, mobility sensor
dyes include, for example, cyanine dyes and derivatives thereof.

[0212] In certain embodiments, the reporter molecule is a dye that
exhibits a change in its spectral properties when specifically bound to a
selectivity component. A nucleic acid, e.g. an aptamer, may be designed
to specifically bind such a dye, for example Malachite Green (see R.
Babendure, et al. (2003) J. Am. Chem. Soc. 125:14716). Such dyes, when in
complex with the nucleic acid or protein that is specific for them,
change their spectral properties. For example, Malachite Green and its
analogs, which is not normally fluorescent, becomes strongly fluorescent
when bound to an aptamer specific for it or an scFv. FIG. 6 depicts the
structure of Malachite Green derivatized with a PEG amine.

[0213] In certain embodiments, the reporter molecule is represented by
structure I, II, or III:

##STR00001##

wherein: the curved lines represent the atoms necessary to complete a
structure selected from one ring, two fused rings, and three fused rings,
each said ring having five or six atoms, and each said ring comprising
carbon atoms and, optionally, no more than two atoms selected from
oxygen, nitrogen and sulfur; D, if present, is

##STR00002##

m is 1, 2, 3 or 4, and for cyanine, oxynol and thiazole orange, m can be
0; X and Y are independently selected from the group consisting of O, S,
and --C(CH3)2--; at least one R1, R2, R3, R4, R5, R6, or R7 is
selected from the group consisting of: a moiety that controls water
solubility and non-specific binding, a moiety that prevents the reporter
molecule from entering the cell through the membrane, a group that
comprises, optionally with a linker, biotin a hapten, a His-tag, or other
moiety to facilitate the process of isolating the selection entity, a
fluorescent label optionally comprising a linker, a photoreactive group,
or a reactive group such as a group containing isothiocyanate,
isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen
substituted pyridine, mono- or di-halogen substituted diazine,
phosphoramidite, maleimnide, aziridine, sulfonyl halide, acid halide,
hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester,
hydrazine, axidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide,
glyoxal, haloacetamido, or aldehyde; further-providing that R1 and R2 may
be joined by a --CHR8--CHR8-- or --BF2-- biradical;
wherein; R8 independently for each occurrence is selected from the
group consisting of hydrogen, amino, quaternary amino, aldehyde, aryl,
hydroxyl, phosphoryl, sulfhydryl, water solubilizing groups, alkyl groups
of twenty-six carbons or less, lipid solubilizing groups, hydrocarbon
solubilizing groups, groups promoting solubility in polar solvents,
groups promoting solubility in nonpolar solvents, and -E-F; and further
providing that any of R1, R2, R3, R4, R5, R6, or R7 may be substituted
with halo, nitro, cyan, --CO2alkyl, --CO2H, --CO2aryl,
NO2, or alkoxy, wherein: F is selected from the group consisting of
hydroxy, protected hydroxy, alkoxy, sulfonate, sulfate, carboxylate, and
lower alkyl substituted amino or quartenary amino; E is spacer group of
formula --(CH2)n- wherein n is an integer from 0-5 inclusively;

[0214] In other embodiments, wherein m=0 in structures I, II, and III, the
following general structures IV, V and VI are afforded:

##STR00003##

wherein: the curved lines represent the atoms necessary to complete a
structure selected from one ring, two fused rings, and three fused rings,
each said ring having five or six atoms, and each said ring comprising
carbon atoms and, optionally, no more than two atoms selected from
oxygen, nitrogen and sulfur; D, if present, is

##STR00004##

X and Y are independently selected from the group consisting of O, S, and
--C(CH3)2--; at least one R1, R2, R3, R4, R5, R6, or R7 is
selected from the group consisting of: a moiety that controls water
solubility and non-specific binding, a moiety that prevents the reporter
molecule from entering the cell through the membrane, a group that
comprises, optionally with a linker, biotin a hapten, a His-tag, or other
moiety to facilitate the process of isolating the selection entity, a
fluorescent label optionally comprising a linker, a photoreactive group,
or a reactive group such as a group containing isothiocyanate,
isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen
substituted pyridine, mono- or di-halogen substituted diazine,
phosphoramidite, maleimnide, aziridine, sulfonyl halide, acid halide,
hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester,
hydrazine, axidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide,
glyoxal, haloacetamido, or aldehyde; further-providing that R1 and R2 may
be joined by a --CHR8--CHR8-- or --BF2-- biradical;
wherein; R8 independently for each occurrence is selected from the
group consisting of hydrogen, amino, quaternary amino, aldehyde, aryl,
hydroxyl, phosphoryl, sulfhydryl, water solubilizing groups, alkyl groups
of twenty-six carbons or less, lipid solubilizing groups, hydrocarbon
solubilizing groups, groups promoting solubility in polar solvents,
groups promoting solubility in nonpolar solvents, and -E-F; and further
providing that any of R1, R2, R3, R4, R5, R6, or R7 may be substituted
with halo, nitro, cyan, --CO2 alkyl, --CO2H, --CO2aryl,
NO2, or alkoxy wherein: F is selected from the group consisting of
hydroxy, protected hydroxy, alkoxy, sulfonate, sulfate, carboxylate, and
lower alkyl substituted amino or quartenary amino; E is spacer group of
formula --(CH2)n- wherein n is an integer from 0-5 inclusively;

[0215] The following are more specific examples of reporter molecules
according to structure I, II, or III:

##STR00005##

[0216] In these structures X and Y are selected from the group consisting
of O. S and --CH(CH3)2--;

Z is selected from the group consisting of O and S; m is an integer
selected from the group consisting of 0, 1, 2, 3 and 4 and, preferably an
integer from 1-3.

[0217] In the above formulas, the number of methine groups determines in
part the excitation color. The cyclic azine structures can also determine
in part the excitation color. Often, higher values of m contribute to
increased luminescence and absorbance. At values of m above 4, the
compound becomes unstable. Thereupon, further luminescence can be
imparted by modifications at the ring structures. When m=2, the
excitation wavelength is about 650 nm and the compound is very
fluorescent. Maximum emission wavelengths are generally 15-100 nm greater
than maximum excitation wavelengths.

[0218] The polymethine chain of the luminescent dyes of this invention may
also contain one or more cyclic chemical groups that form bridges between
two or more of the carbon atoms of the polymethine chain. These bridges
might serve to increase the chemical or photostability of the dye and
might be used to alter the absorption and emission wavelength of the dye
or change its extinction coefficient or quantum yield.

[0219] Improved solubility properties may be obtained by this
modification.

In certain embodiments, the reporter molecule is represented by structure
VII:

##STR00006##

wherein:

W is N or C(R1);

X is C(R2)2;

Y is C(R3)2;

Z is NR1, O, or S;

[0220] at least one R1, R2, or R3 is selected from the group consisting
of: a moiety that controls water solubility and non-specific binding, a
moiety that prevents the reporter molecule from entering the cell through
the membrane, a group that comprises, optionally with a linker, biotin, a
hapten, a His-tag, or other moiety to facilitate the process of isolating
the selection entity, a fluorescent label optionally comprising a linker,
a photoreactive group, or a reactive group such as a group containing
isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono-
or di-halogen substituted pyridine, mono- or di-halogen substituted
diazine, phosphoramidite, maleimnide, aziridine, sulfonyl halide, acid
halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido
ester, hydrazine, axidonitrophenyl, azide, 3-(2-pyridyl
dithio)-proprionamide, glyoxal, haloacetamido, or aldehyde; further
providing that two R3 taken together may form O, S, NR1, or N+(R1)2;
or two R3 along with R2 may form

##STR00007##

wherein V is O, S, NR1, or N+(R1)2; and further providing that any
of R1, R2, or R3 may be substituted with halo, nitro, cyano,
--CO2alkyl, --CO2H, --CO2aryl, NO2, or alkoxy.

[0221] The following are more specific examples of reporter molecules
according to structure VII:

##STR00008##

[0222] In certain embodiments, the reporter molecule is represented by
structure VIII:

##STR00009##

wherein: at least one R1 is selected from the group consisting of: a
moiety that controls water solubility and non-specific binding, a moiety
that prevents the reporter molecule from entering the cell through the
membrane, a group that comprises, optionally with a linker, biotin, a
hapten, a His-tag, or other moiety to facilitate the process of isolating
the selection entity, a fluorescent label optionally comprising a linker,
a photoreactive group, or a reactive group such as a group containing
isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono-
or di-halogen substituted pyridine, mono- or di-halogen substituted
diazine, phosphoramidite, maleimnide, aziridine, sulfonyl halide, acid
halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido
ester, hydrazine, axidonitrophenyl, azide, 3-(2-pyridyl
dithio)-proprionamide, glyoxal, haloacetamido, or aldehyde; further
providing that any two adjacent R1, in certain embodiments, may be joined
to form a fused aromatic ring; and further providing that R1, in certain
embodiments, may be substituted with halo, nitro, cyan, --CO2alkyl,
--CO2H, --CO2aryl, NO2, or alkoxy.

[0223] In certain embodiments, at least one, preferably only one, and
possibly two or more of either R1, R2, R3, R4, R5, R6 and R7 groups in
each of the foregoing molecules is or contains a reactive group
covalently reactive with amine, protected or unprotected hydroxy or
sulfhydryl nucleophiles for attaching the dye to the labeled component.
For certain reagents, at least one of said RI, R2, R3, R4, R5, R6 and R7
groups on each molecule may also be a group that increases the solubility
of the chromophore, or affects the selectivity of labeling of the labeled
component or affects the position of labeling of the labeled component by
the dye.

wherein at least one of Q or W is a leaving group such as I, Br or Cl and
n is an integer from 0 to 4.

[0226] Specific examples of R1, R2, R3, R4, R5, R6 and R7 groups that are
especially useful for labeling components with available sulfhydryls
which can be used for labeling selectivity components in a two-step
process are the following:

##STR00012##

wherein Q is a leaving group such as I or Br, and wherein n is an integer
from 0 to 4. Specific examples of R1, R2, R3, R4, R5, R6 and R7 groups
that are especially useful for labeling components by light-activated
cross linking include:

##STR00013##

[0227] For the purpose of increasing water solubility or reducing unwanted
nonspecific binding of the labeled component to inappropriate components
in the sample or to reduce the interactions between two or more reactive
chromophores on the labeled component which might lead to quenching of
fluorescence, the R1, R2; R3, R4, R5, R6 and R7 groups can be selected
from the well known polar and electrically charged chemical groups.

[0228] In certain embodiments of the invention, the reporter molecule is
represented by structure I, II, III, IV, V, VI, VII or VIII and the
accompanying definitions, and is a pH sensitive reporter molecule.

[0229] In certain embodiments of the invention, the reporter molecule is
represented by structure I, II, III, IV, V, VI, VII or VIII and the
accompanying definitions, and is a polarity sensitive reporter molecule.

[0230] In certain embodiments of the invention, the reporter molecule is
represented by structure I, II, III, IV, V, VI, VII or VIII and the
accompanying definitions, and is a microviscosity reporter molecule.

[0231] In certain embodiments of the invention, the reporter molecule is
represented by structure I, II, III, IV, V, VI, VII or VIII and the
accompanying definitions, and is a mobility sensor reporter molecule.

[0232] In various other embodiments, the reporter is a of the type class
IV: wherein the dye has the general structure

A-B=A'

[0233] wherein A is selected from

##STR00014##

[0234] wherein A' is selected from

##STR00015##

[0235] wherein R1, R2, R3, R4, R5 and R6 are selected from the group
consisting of: a moiety that controls water solubility and non-specific
binding, a moiety that prevents the dye from entering a cell through a
membrane, a group that comprises, optionally with a linker, biotin, a
hapten, a His-tag, or a moiety to facilitate isolation of the ligand, a
fluorescent label optionally comprising a linker, a photoreactive group,
a reactive containing isothiocyanate, isocyanate, monochlorotriazine,
dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or
di-halogen substituted diazine, phosphoramidite, maleimnide, aziridine,
sulfonyl halide, acid halide, hydroxysuccinimide ester,
hydroxysulfosuccinimide ester, imido ester, hydrazine, axidonitrophenyl,
azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal, haloacetamido, or
aldehyde, and, R1 and R2 may be joined by a --CHR8--CHR8-- or
--BF2-- biradical,

[0237] wherein F is selected from the group consisting of hydroxy,
protected hydroxy, alkoxy, sulfonate, sulfate, carboxylate, and alkyl
substituted amino or quartenary amino and E is spacer group of formula
--(CH2)n- wherein n is an integer from 0-5 inclusively; and,

[0241] Z is selected from is H, C1-C18 alkyl, C1-C18
substituted alkyl, cyclic and heterocyclic having from one ring, two
fused rings, and three fused rings, each said ring having three to six
atoms, and each said ring comprising carbon atoms and from zero to two
atoms selected from oxygen, nitrogen and sulfur and containing zero to 1
oxygen, nitrogen or sulfur substituents attached; and,

[0242] Z' is selected from H, C1-C18 alkyl, C1-C18
substituted alkyl, cyclic and heterocyclic having from one ring, two
fused rings, and three fused rings, each said ring having three to six
atoms, and each said ring comprising carbon atoms and from zero to two
atoms selected from oxygen, nitrogen and sulfur, A, and A'.

[0243] In various embodiments, the spectral change of the sensor dye upon
interaction of the selectivity component and a target molecule may
include, for example, a shift in absorption wavelength, a shift in
emission wavelength, a change in quantum yield, a change in polarization
of the dye molecule, and/or a change in fluorescence intensity. Any
method suitable for detecting the spectral change associated with a given
sensor dye may be used in accordance with the inventions. In exemplary
embodiments, suitable instruments for detection of a sensor dye spectral
change, include, for example, fluorescent spectrometers, filter
fluorometers, microarray readers, optical fiber sensor readers,
epifluorescence microscopes, confocal laser scanning microscopes, two
photon excitation microscopes, and flow cytometers.

[0244] In variant embodiments, the reporter molecule may be associated
with the selectivity component or the target molecule. In exemplary
embodiments, the reporter molecule is covalently attached to the
selectivity component. The reporter molecule may be covalently attached
to the selectivity component using standard techniques. In certain
embodiments the reporter molecule may be directly attached to the
selectivity component by forming a chemical bond between one or more
reactive groups on the two molecules. In an exemplary embodiment, a thiol
reactive reporter molecule is attached to a cysteine residue (or other
thiol containing molecule) on the selectivity component. Alternatively,
the reporter molecule may be attached to the selectivity component via an
amino group on the selectivity component molecule. In other embodiments,
the reporter molecule may be attached to the selectivity component via a
linker group. Suitable linkers that may be used in accordance with the
inventions include, for example, chemical groups, an amino acid or chain
of two or more amino acids, a nucleotide or chain of two or more
polynucleotides, polymer chains, and polysaccharides. In exemplary
embodiments, the reporter molecule is attached to the selectivity
component using a linker having a maleimide moiety. Linkers may be
homofunctional (containing reactive groups of the same type),
heterofunctional (containing different reactive groups), or photoreactive
(containing groups that become reactive on illumination). A variety of
photoreactive groups are known, for example, groups in the nitrene
family.

[0245] In various embodiments, one or more reporter molecules may be
attached at one or more locations on the selectivity component. For
example, two or more molecules of the same reporter may be attached at
different locations on a single selectivity component molecule.
Alternatively, two or more different reporter molecules may be attached
at different locations on a single selectivity component molecule. In
exemplary embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more reporter
molecules are attached at different sites on the selectivity component.
The one or more reporter molecules may be attached to the selectivity
component so as to maintain the activity of the reporter molecule and the
selectivity component.

[0246] In certain embodiments, the location of the reporter molecule is
optimized to permit exposure of the reporter molecule to changes in the
microenvironment upon interaction of the selectivity component with a
target molecule while maintaining the ability of the selectivity
component to interact with the target molecule. In exemplary embodiments,
the reporter molecule is attached to the selectivity component in spatial
proximity to the target binding site without affecting the ability of the
selectivity component to interact with the target molecule.

[0247] In certain embodiments, the reporter molecule further comprises a
moiety that is specific for the selectivity component. For example, the
reporter molecule may be linked to a substrate, a hapten, etc. that is
specific for the selectivity component if it is an enzyme, hapten-binding
protein, etc. The reporter molecule may be covalently attached to the
moiety using standard techniques. In certain embodiments the reporter
molecule may be directly attached to the moiety by forming a chemical
bond between one or more reactive groups on the two molecules. In other
embodiments, the reporter molecule may be attached to the moiety via a
linker group. Suitable linkers that may be used in accordance with the
inventions include, for example, chemical groups, an amino acid or chain
of two or more amino acids, a nucleotide or chain of two or more
polynucleotides, polymer chains, and polysaccharides. Linkers may be
homofunctional (containing reactive groups of the same type),
heterofunctional (containing different reactive groups), or photoreactive
(containing groups that become reactive on illumination).

[0248] 5. Exemplary Uses

[0249] The biosensors of the invention may be used to detect and/or
quantitate analytes in any solid, liquid or gas sample, as well as in any
cell or tissue or organism of interest. In various exemplary embodiments,
the biosensors of the invention may be used for a variety of diagnostic
and/or research applications, including, for example, monitoring the
development of engineered tissues, in vivo monitoring of analytes of
interest (including polynucleotides, polypeptides, hormones, lipids,
carbohydrates, and small inorganic and organic compounds and drugs) using
injectable free biosensors or implants functionalized with one or more
biosensors, biological research (including developmental biology, cell
biology, neurobiology, immunology, and physiology); detection of
microbial, viral and botanical polynucleotides or polypeptides, drug
discovery, medical diagnostic testing, environmental detection (including
detection of hazardous substances/hazardous wastes, environmental
pollutants, chemical and biological warfare agents, detection of
agricultural diseases, pests and pesticides and space exploration),
monitoring of food freshness and/or contamination, food additives, and
food production and processing streams, monitoring chemical and
biological products and contaminants, and monitoring industrial and
chemical production and processing streams.

[0254] In one embodiment, the biosensors described herein may be used for
in vitro and/or in vivo monitoring of analytes of interest. The
biosensors may be injected or otherwise administered to a patient as free
molecules or may be immobilized onto a surface before introduction into a
patient. When administered. as free molecules, the biosensors may be used
to detect analytes of interest in both interstitial spaces and inside
cells. For detection of analytes inside of cells, the selectivity
component may be modified, as described above, with a tag that
facilitates translocation across cellular membranes. Alternatively, the
selectivity components may be introduced into cells using liposome
delivery methods or mechanical techniques such as direct injection or
ballistic-based particle delivery systems (see for example, U.S. Pat. No.
6,110,490). In other embodiments, the biosensors may be immobilized onto
a surface (including, for example, a bead, chip, plate, slide, strip,
sheet, film, block, plug, medical device, surgical instrument, diagnostic
instrument, drug delivery device, prosthetic implant or other-structure)
and then introduced into a patient, for example, by surgical
implantation. In exemplary embodiments, the biosensors are immobilized
onto the surface of an implant, such as an artificial or replacement
organ, joint, bone, or other implant. The biosensors of the invention may
also be immobilized onto particles, optical fibers, and polymer scaffolds
used for tissue engineering. For example, one or more biosensors may be
immobilized onto a fiber optic probe for precise positioning in a tissue.
The fiber optic then provides the pathway for excitation light to the
sensor tip and the fluorescence signal back to the photodetection system.
In still other embodiments, at least the selectivity component of the
biosensor is transfected in cell or other host organism of interest and
expressed within the cell or other host organism of interest.

[0255] In each of the various embodiments of the invention, a single
biosensor may be used for detection of a single target molecule or two or
more biosensors may be used simultaneously for detection of two or more
target molecules. For example, 2, 5, 10, 20, 50, 100, 1000, or more
different selectivity components may be used simultaneously for detection
of multiple targets.

[0256] When using multiple selectivity components simultaneously, each
selectivity component may be attached to a different reporter molecule to
permit individual detection of target binding to each selectivity
component. Alternatively, a dual detection system may be used where two
or more selectivity components may be attached to the same reporter
molecule (for example, the same sensor dye) and be identified based on a
second detectable signal. For example, selectivity components having
different target specificities but containing the same sensor dye may be
distinguished based on the signal from a color coded particle to which it
is attached. The readout for each selectivity component involves
detection of the signal from the sensor dye, indicating association with
the target molecule, and detection of the signal from the color coded
particle, permitting identification of the selectivity component. In an
exemplary embodiment, a panel of biosensors may be attached to a variety
of color coded particles to form a suspension array (Luminex Corporation,
Austin, Tex.). The mixture of coded particles associated with the
biosensors of the invention may be mixed with a biological sample or
administered to a patient. Flow cytometry or microdialysis may then be
used to measure the signal from the sensor dye and to detect the color
code for each particle. In various embodiments, the identification signal
may be from a color coded particle or a second reporter molecule,
including, for example, chemiluminescent, fluorescent, or other optical
molecules, affinity tags, and radioactive molecules.

[0257] In other embodiments, one or more biosensors of the invention may
be immobilized onto a three dimensional surface suitable for implantation
into a patient. The implant allows monitoring of one or more analytes of
interest in a three dimensional space, such as, for example, the spaces
between tissues in a patient.

[0258] In other embodiments, the biosensors of the invention may be
exposed to a test sample. Any test sample suspected of containing the
target may be used, including, but not limited to, tissue samples such as
biopsy samples and biological fluids such as blood, sputum, urine and
semen samples, bacterial cultures, soil samples, food samples, cell
cultures, etc. The target may be of any origin, including animal, plant
or microbiological (e.g., viral, prokaryotic, and eukaryotic organisms,
including bacterial, protozoal, and fungal, etc.) depending on the
particular purpose of the test. Examples include surgical specimens,
specimens used for medical diagnostics, specimens used for genetic
testing, environmental specimens, cell culture specimens, food specimens,
dental specimens and veterinary specimens. The sample may be processed or
purified prior to exposure to the biosensor(s) in accordance with
techniques known or apparent to those skilled in the art.

[0259] In other embodiments, the biosensors of the invention may be used
to detect bacteria and eucarya in food, beverages, water, pharmaceutical
products, personal care products, dairy products or environmental
samples. The biosensors of the invention are also useful for the analysis
of raw materials, equipment, products or processes used to manufacture or
store food, beverages, water, pharmaceutical products, personal care
products, dairy products or environmental samples.

[0260] Alternatively, the biosensors of the invention may be used to
diagnose a condition of medical interest. For example the methods, kits
and compositions of this invention will be particularly useful for the
analysis of clinical specimens or equipment, fixtures or products used to
treat humans or animals. In one preferred embodiment, the assay may be
used to detect a target sequence which is specific for a genetically
based disease or is specific for a predisposition to a genetically based
disease. Non-limiting examples of diseases include, beta-thalassemia,
sickle cell anemia, Factor-V Leiden, cystic fibrosis and cancer related
targets such as p53, p 10, BRC-1 and BRC-2. In still another embodiment,
the target sequence may be related to a chromosomal DNA, wherein the
detection, identification or quantitation of the target sequence can be
used in relation to forensic techniques such as prenatal screening,
paternity testing, identity confirmation or crime investigation.

[0261] In still other embodiments, the methods of the invention include
the analysis or manipulation of plants and genetic materials derived
therefrom as well as bio-warfare reagents. Biosensors of the invention
will also be useful in diagnostic applications, in screening compounds
for leads which might exhibit therapeutic utility (e.g. drug development)
or in screening samples for factors useful in monitoring patients for
susceptibility to adverse drug interactions (e.g. pharmacogenomics).

[0262] In certain embodiments, the biosensors of the invention, or nucleic
acids encoding them, may be formulated into a pharmaceutical composition
comprising one or more biosensors and a pharmaceutically acceptable
carrier, adjuvant, or vehicle. The term "pharmaceutically acceptable
carrier" refers to a carrier(s) that is "acceptable" in the sense of
being compatible with the other ingredients of a composition and not
deleterious to the recipient thereof. Methods of making and using such
pharmaceutical compositions are also included in the invention. The
pharmaceutical compositions of the invention can be administered orally,
parenterally, by inhalation spray, topically, rectally, nasally,
buccally, vaginally, or via an implanted reservoir. The term "parenteral"
as used herein includes subcutaneous, intracutaneous, intravenous,
intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal,
intralesional, and intracranial injection or infusion techniques. In
other embodiments, the invention contemplates kits including one or more
biosensors of the invention, and other subject materials, and optionally
instructions for their use. Uses for such kits include, for example,
environmental and/or biological monitoring or diagnostic applications.

[0263] A biosensor, or an isolated, purified biosensor, comprising the
selectivity component may have at least about 85% sequence identity with
SEQ ID NO:1 through 72. The selectivity component may reversibly bind
either a monomethin cyanine dye, or Malachite Green, or an analog
thereof. A host cell may express the biosensor. Further, a vector may be
comprised of a nucleic acid sequence having at least about 85%, and
preferably 95%, sequence identity to a polynucleotide encoding a protein
with SEQ ID NO:1 through 72. A host cell may comprise the vector.

EXAMPLES

[0264] The invention now being generally described, it will be more
readily understood by reference to the following examples, which are
included merely for purposes of illustration of certain aspects and
embodiments of the present invention, and are not intended to limit the
invention.

[0265] Provided are several single chain antibody (scFv) based sensors
that comprise amino acid sequences that lead to specific binding of
certain monomethin cyanine dyes (TO1 and its analogs (the structure of
TO1 derivatized with PEG amine is shown in FIG. 7) and Malachite Green
(and its analogs) in a way that produces a large increase in fluorescence
of the dye (a "fluorogen") when it is in the bound state.

Example 1

scFv-Based Sensors that Reversibly Bind Fluorescent Dyes

[0266] In one experiment, an scFv that reversibly binds noncovalently the
dye TO1 ("scFv1") was produced. See, for example, SEQ ID NO:1. A
2700-fold increase in fluorescence was detected (by methods described
below) when the TO1 binds to the scFv. This sequence was used in
subsequent work to produce additional scFvs that bind to TO1 with a
stronger binding affinity and have enhanced fluorescence. In other
experiments, scFvs were developed that bind certain derivatives of
malachite green where the noncovalent binding is strong and again a large
increase in fluorescence is produced upon binding of the dye to the scFv.
See, FIGS. 9, 10A and 10B.

1A

[0267] The genetic sequence of this scFv may be inserted into genes for
other proteins so that the expressed fusion protein will contain the
dye-binding scFv. For example, a cell surface membrane protein was
labeled with scFv1 by genetic methods and expressed on the cell surface
of cultured mammalian cells. When the relatively non-fluorescent (in
water or buffer) dye TO1 was added to cell suspensions containing the
protein-scFv, a fraction of the dye binds to the scFv with a large (more
than 2500×) fluorescence increase upon binding and thereby produced
a fluorescent label on the protein of interest on the membrane surface.
Thus, the appearance of fluorescence when the scFv and the dye are both
present is very rapid. Thus, TO1 and the corresponding TO1 scFv may be
used to detect protein expression of the scFv-fusion protein on the
surface of the cell where the scFv will have access to the membrane
impermeant dye that has been added to the culture medium. Other proteins
may be labeled with the scFv and if the labeled protein is present inside
the cell, a membrane permeant fluorogen may be used to detect the
presence of that particular scFv fusion protein. The presence of the scFv
can be used to ascertain the amount, expression, degradation, or location
of the fusion protein.

[0268] In one experiment, yeast FAPs were fused via AGA2 to the AGA1-AGA2
complex, which is directed to the outer leaflet of the plasma membrane by
a C-terminal glycosylphosphatidylinositol (GPI) anchor before insertion
into the cell wall. Live cell imaging using fluorescent proteins fused to
GPIs or GPI-anchored proteins is useful for studying organization and
function of membrane proteins, including signaling receptors and cell
adhesion molecules, but these constructs may also label cell structures
involved in biosynthesis, secretion and degradation. Dynamic imaging of
lipid rafts and other surface features would benefit by confining
fluorescence to proteins anchored to the outer leaflet. Whereas methods
such as total internal reflection fluorescence microscopy have evolved to
allow selective observation, there are no methods for selective labeling
and homogenous detection. To illustrate such labeling and detection, an
MG FAP and an identically anchored AGA2-GFP fusion protein was imaged.
The MG FAP and the GFP were visualized on the extracellular surface, but
intracellular structures, many with the morphology expected of vacuoles
and nuclear membranes (endoplasmic reticulum), were visualized only by
the GFP. To explore fluorogenic labeling of mammalian cell surface
proteins, selected TO1 and MG FAPs were fused to the N terminus of
platelet-derived growth factor receptor (PDGFR) transmembrane domain. The
TO1 and MG FAPs were than expressed stably in NIH3T3 mouse fibroblasts
and in M21 human renal carcinoma cells. In each case the transfected
cells exhibited strong surface fluorescence when exposed to low
concentrations of TO1-2p or MG-11p fluorogen. No significant
intracellular fluorescence was observed under these experimental
conditions; TO1-2p and MG-11p controls did not enter living NIH3T3 cells.
It is noteworthy that little or no photobleaching of cell surface
fluorescence was observed. Separate experiments showed that TO1-2p FAPs
resisted bleaching about as well as EGFP and that MG FAPs were even more
bleach resistant (FIG. 16). Fluorescence signal of TO1-2p FAPs decayed to
a TO1-2p concentration dependent steady state, which suggests without
being bound by theory, that rapid exchange of fluorogen (and/or fluorogen
photoproducts) between the solution and the FAP itself was effectively
buffering the system against photobleaching.

[0269] For MG FAPs, other mechanisms may contribute, such as loose
sequestration of dark fluorogen on the plasma membrane outer surface. In
aqueous solution mobile fluorogens such as MG or TO 1 show almost no
photoreactivity, but under illumination MG conjugated to an antibody
generates reactive oxygen species at a rate similar to GFP, sufficient to
be phototoxic under continuous or intense excitation. Phototoxicity
correlates with photobleaching, suggesting that MG and TO1 FAPs generated
reactive oxygen species at GFP-like rates. MG has also been used as an
antifungal agent; at experimental concentrations the MG derivatives
studied here had little or no effect on yeast growth (FIG. 17). Cell
surface-exposed FAPs visualized with a membrane-impermeant fluorogen were
seen at the plasma membrane only. When exposed to a membrane-permeant
fluorogen, however, these same cells showed additional fluorescence
within elements of the secretory apparatus, including the nuclear
endoplasmic reticulum and the Golgi. This result suggests that permeant
fluorogens can be used to visualize FAPs shortly after cotranslational
insertion into the lumen, and thus potentially report protein folding in
near real time. Permeant fluorogens can be added and withdrawn at will,
facilitating development of pulse-chase and other approaches to studying
secretory and endocytic pathways. When incorporated into fusion proteins,
FAP domains provided a reporter of protein location and abundance in time
and space. Fluorescence signal was generated only upon addition of a
second component (the fluorogen); in this respect FAPs resemble the
site-specific chemical labeling systems. However, all chemical labeling
systems require additional manipulation such as enzymatic conjugation
steps or washes to reduce background signal, whereas FAPs can be
visualized directly after fluorogen addition on a time scale of seconds
(on the cell surface) to minutes (within the secretory apparatus).
Fluorescence visualization can also be spatially controlled by the
appropriate choice of fluorogen, enabling one to selectively observe
fusion proteins at particular cellular locations. Multicolor imaging of
spectrally and antigenically distinct FAPs co-expressed within a cell
will greatly enhance the usefulness of different fluorogens to
dynamically monitor complex cellular functions. ScFv-based FAPs contain
internal disulfide linkages and are currently adapted for use only in
nonreducing environments, mainly the cell surface and secretory
apparatus. FAP/fluorogens thus complement the biarsenical system, which
is generally limited to intracellular reducing environments, primarily
the cytoplasmic and nuclear compartments. However, it has been shown that
functional scFvs can be expressed cytoplasmically in a disulfide-free
format in yeast and mammalian cells, and future developments of scFv and
other FAP platforms will address these intracellular compartments.

1B

[0270] Additionally, the genetic sequence of an scFv may be used to create
molecular biosensors. In one example, the initial sequence of an scFv is
modified to make the binding of a reporter molecule by the scFv sensitive
to the presence of other molecular interactions with the scFv. Such
interactions include contact with another protein or peptide. Such
interactions can involve contact with a kinase or phosphatase or other
covalent modification that alters an amino acid of the scFv to produce a
change in fluorogen fluorescence. The interaction can produce a steric
change or allosteric change near the reporter group on the sensor that
produces the fluorescence signal. The interaction can alter a charged
amino acid side chain or an ionizable group on a side chain or hydrogen
bonding group or a non-polar group near the reporter that produces a
fluorescence signal.

1C

[0271] The TO1 scFv was created using a large PNNL Yeast Display library
comprised of cells that each express on its surface on of an estimated
109 different sequence variations in the heavy and light variable
regions that constitute the displayed scFv proteins. The TO1-binding scFv
was isolated in two steps. First, a TO1 dye linked via a PEG polymer to a
biotin was used in a magnetic bead separation procedure to isolate a
population of cells that was enriched for cells that bind in variable
degrees to the TO1 dye. In a second step, flow cytometry experiments were
carried out to select for cells that bind the dye and make it highly
fluorescent. One of the highly fluorescent cells was sorted and cloned.
This yeast cell was the source of the scFv. Yeast plasmid DNA encoding
this scFv clone was amplified by PCR methods, and then sequenced. Flow
cytometry data has demonstrated the successful cloning of this highly
fluorescent TO1-binding scFv. Similarly for malachite green.

1D

[0272] It is possible to engineer and select other proteins that are
capable of binding to the fluorescent reporters of this invention such
that they also would function as biosensors in a way similar to the scFv1
of this example. Such engineered proteins that can be used as biosensors
we call Fluorescent Binding Protein (FBP) sensors. Examples of such
non-immunological binding proteins engineered to bind small-to-medium
molecular weight molecules are described by Bintz et al. (2005) Nature
Biotech. 23:1257-1268.

Example 2

Reporter Molecule-Localizing System Based on SAb scFv Technology

[0273] In this example, a combination of reagents with which the location
of the reporter molecule can be controlled within a cell by genetic
methods

[0274] In biological research and pharmaceutical drug discovery it is
useful to be able to target specific reporter molecules to certain
regions or structures within a cell. Examples of such specific reporter
molecules include regulatory metabolites such as small peptides, growth
factors, and inhibitory RNAs. Also such reporter molecules s may include
synthetic and natural molecules that modify cellular behavior such as
those often used and developed by the pharmaceutical industry. Further,
such reporter molecules might also include fluorescent or light absorbing
molecules that provide a signal for targeting of a protein within a cell
or that are sensitive to a physiological property of the cell such as
membrane potential, ion concentration or enzyme activity. In other words,
the reporter molecules are used to modulate or perturb the activity of
specific proteins, pathways and networks within cells or to measure
biochemical, structural and physiological properties of cells. The
reporter molecules allow control of targeting of the region of a cell or
tissue where the perturbation or measurement occurs.

[0275] Many types of reporter molecules diffuse into cells and perturb or
report from many regions of a cell where the probe non-specifically
associates. The targeting in the reporter molecules may be controlled by
bringing together two molecular entities within a cell or on its surface
or in a tissue. One such entity may comprise the reporter molecule, a
hapten and a water soluble linker that separates the probe and the
hapten. The hapten is a molecule with which antibodies that bind the
molecule can be generated by known procedures. This three part structure
is shown on the right hand side of FIG. 3. Such a reporter might be a
voltage sensitive dye, a calcium indicator, a pH indicator, ion
indicator, polarity indicator, mechanical stress indicator or an
indicator of some other physiological or molecular process occurring in
the cell.

[0276] The second entity required for targeting the probe in a cell
consists of a selectivity component, e.g., a hapten-binding antibody,
specifically a scFv that may be genetically fused to a "protein of
interest" within the cell. The protein of interest serves the purpose of
localizing the reporter molecule-binding scFv, and thus the reporter
molecule after it has been incorporated in the cell or tissue, to a
specific region of interest within or on a cell. The protein of interest
and the scFv are illustrated on the left hand side of FIG. 3.

[0277] The process of targeting the reporter molecule within the cell
takes place in several steps: (1) first, a hapten-linker-reporter
molecule must be synthesized, then (2) a hapten-binding scFv must be
created. This may be done, for example, by yeast selection procedures
developed by Wittrup, et al. (Methods Enzvmol. (2000) 328:430-33; Proc.
Natl. Acad. Sci. USA (2000) 97:10701-5; Nucleic Acids Res (2004) 32:e36).
(3) The cell of interest must be transfected with a gene that codes for
the protein of interest fused to the hapten-binding protein. (4) The
hapten-linker-reporter molecule must be incorporated into the cell by
diffusion through the membrane or microinjection or by another means. The
result of this process will be noncovalent placement of the reporter
molecule into the protein of interest that may be in the nucleus, or an
organelle, in the cytoplasm, on the internal surface of the membrane, or
in a specific location of a tissue. By using a photoreactive group or a
reactive functional group the reporter moiety may be covalently attached
to the protein of interest.

Several variations are possible:

[0278] Variation 1: Non-Covalent Binding of the Hapten-Linker-Reporter
Molecule to the scFv.

[0279] The affinity of this binding depends on the structure of the scFv
and the hapten. In some cases, it is desirable to have relatively weak
binding so that the reporter molecule can experience the targeted protein
of interest but also other regions of the cell. In other cases, it is
desirable to have strong binding so that the probe is predominantly at
the scFv. In this case, there would be less non-specific binding of the
hapten-linker-reporter molecule to other regions of the tissue and the
modulating or measuring capabilities of the reporter molecule would be
targeted mainly to the scFv-protein of interest region. There would
therefore be a better signal-to-noise in the experiment. The binding
constant of the hapten to the antibody (often expressed as a dissociation
constant, Kd) can be adjusted by the procedures used to create the
hapten-binding scFv. By yeast selection procedures and by a process
called affinity maturation (where the scFv is genetically mutated and
further selection is carried out) it is possible to considerably decrease
Kd (increase the binding affinity).

[0280] Variation 2: Covalent Linkage of the Hapten-Linker-Reporter
Molecule to the scFv Using Light.

[0281] In this case, the hapten is modified to contain a specific reactive
group or a photo-reactive group that will cause the scFv binding group to
permanently and covalently associate with the targeting scFv. The
photoreactive group could be placed adjacent to the hapten or could be
structurally part of the hapten. A scFv that binds the hapten or the
modified hapten may be created. The photoreactive hapten is called a
"photohapten." Illumination of the cell or tissue containing the
photohapten-linker-probe and the scFv-protein of interest would cause
covalent linkage of the photohapten groups that are within the scFv
binding site to a region of the scFv. A potential advantage of this
approach is that excess non-scFv associating hapten-linker-reporter
molecule could be washed out, so long as it does not photochemically
react with other cellular structures. Any of a variety of known
photoreactive groups may be used, such as those of the nitrene family.
Several examples of reporter molecules are shown in FIG. 4. The compounds
have sites for attachment to a linker and thus to a reporter molecule.

[0282] Variation 3: Photo-Controlled Reversible Binding of the Reporter
Molecule to the scFv.

The binding of the reporter molecule may be reversibly controlled with
light by a photoreaction upon illumination of the scFv binding group
chromophores that alters its molecular conformation and thereby its
affinity for the binding site of the targeting scFv. There are known
organic chromophores that undergo conformational changes upon
illumination. Stilbenes and azo-compounds undergo cis-trans
isomerization. Spiranes undergo ring opening as shown in FIG. 5.

[0283] In producing such a reversible system, the scFv may be generated
using the predominant species of the equilibrium at room temperature,
which for example is the spiro form of the compound in FIG. 5 that is
>99% in the absence of UV light. When the reagent system is in the
cell or tissue, the reporter molecule would then be released from the
scFv-protein of interest by illuminating the sample with UV light. For
recapture of the reporter molecule by the scFv a longer wavelength of
illumination that excites the mero form may be used, or the reaction may
be incubated to achieve thermal re-equilibrium of the reaction to favor
the spiro form. Such a reagent system may be used to transient release of
metabolic factors or drugs that modify regulatory pathways in cells and
tissues. A variety of photoreversible chromophores are known in the art
and have been recently described in Sakata, et al., Proc. Natl. Acad.
Sci. USA (2005) 102(13):4759-4764.

[0284] Variation 4: Photo-Release of Targeted Reporter Molecule

Photo-uncaging of cell and tissue modulating agents have been widely used
by biologists and biophysicists. Generally, an inactive form of a
modulating agent or reporter molecule is illuminated to release an active
form into the illuminated region. In this case, the modulating agent may
be active, but targeted to the scFv-containing region of the cell or
tissue. Illumination would release the material, allowing it to diffuse
and produce effects elsewhere in the cell or tissue. Photo-uncaging
chemistries are known (Curtin, et al. Photochemistry and Photobiology
(2005) 81:641-648) and may be inserted at a convenient site between the
hapten and the reporter molecule.

[0285] Currently, there is no way to target such reagents to specific
types of cells in a heterogeneous mixture of cells. Through genetic
targeting, the reporter molecule could be sequestered and remain caged in
a defined region of the cell through the genetic targeting of the scFv to
the cell of interest. Also, addition of the "caged reporter molecule" to
the cells would allow the targeted scFv containing cell to specifically
and strongly bind the caged reporter molecule. Washing the cells would
remove the caged reporter molecule from cells that are not of interest
and illumination would produce release of the reporter molecule only in
the cells of interest.

[0286] In this invention scFv-based binding proteins offer one type of
fluorescent binding protein that can be used to create biosensors as in
Example 3. As mentioned earlier there are other FBPs that could also be
engineered to create sensors.

Example 3

A Biosensor for Protein-Protein Interaction Based on scFv Technology

[0287] Protein-protein interactions are widely used by living cells to
regulate important pathways controlling cell growth and behavior. There
are examples in the literature of the study of protein-protein
interactions by protein complementation (TK Kerppola (2006) Nature
Methods 3:969). The protein-protein interaction event is detected by
attaching the two cleaved parts of a sensor protein to the two proteins
whose interaction is to be detected. When the interaction takes place the
cleaved parts of the sensor protein complement (interact with) one
another to form a functional protein. The known examples in the
literature do not include the use of fluorescent reporter binding scFvs
described above.

[0288] This example involves the use of the heavy (H) and light (L)
fragments of single chain antibodies (scFvs) that have been selected to
bind fluorescent reporter molecules. Once a ScFv with appropriate
fluorogen has been obtained and the genetic sequence of the ScFv
determined, molecular biology methods are available to modify genetic
sequences into the ScFv gene at certain locations. The modified genetic
sequences can be inserted into yeast expression systems to produce
secreted protein or to produce surface displayed ScFv that correspond to
the genetic modifications. It is further possible to eliminate the
genetic sequence corresponding to the short polypeptide linker that holds
the H and L chains of the selected ScFv together. Thus it is possible to
obtain independently the H and L halves of the original ScFv. It is
further possible to attach the genetic sequence of the H chain to a
protein, for example, P1, and the L chain to a second protein, P2 to
obtain two DNA sequences that will give fusion proteins upon protein
expression. The goal is to investigate whether P1 and P2 interact within
or outside cells. The genetic sequences for the P1-H and P2-L fusion
proteins can then be transfected into living cells where the cells will
produce the two fusion protein products. In this example when the P1-H
and P2-L are diffusing independently in the interior of a cell, neither
the H nor the L fragments alone will bind the fluorogen (that bound with
a fluorescence increase to the original ScFv from which the H and L
halves were obtained) to produce a significant fluorescence signal.
However, when P1 and P2 interact, the H and L components will be brought
into close proximity and will interact as well to form the original
combining site that will bind to the fluorogen. If this is the case, a
fluorescence signal will occur on interaction of P1 and P2.

[0289] The protein-protein biosensor of this example has advantages of
quick fluorescence response, good sensitivity and relative reversibility.
It is possible to use fragments of other FBPs to create protein-protein
interaction sensors similar to the one described above.

[0290] In this experiment, it was demonstrated that fluorescent proteins
for live cell applications could be created. Eight unique FBPs that
elicit intense fluorescence from otherwise dark dyes were isolated by
screening a yeast display library of human single chain antibodies
(scFvs) using derivatives of thiazole orange (TO) and malachite green
(MG). When displayed on yeast or mammalian cell surfaces, these FBPs bind
their fluorogens with nanomolar affinity, increasing their respective
green or red fluorescence by several thousand-fold to brightness levels
similar to that of enhanced green fluorescent protein. Significant
spectral variation is generated within the family of malachite green FBPs
by use of different proteins and chemically modified fluorogens. These
diverse FBPs and fluorogens provide opportunities to create new classes
of biosensors and new homogeneous cell-based assays.

[0291] These studies were aimed at creating a new class of protein/dye
reporters whose spectral properties are determined by the interplay of a
protein moiety (a Fluorescent Binding Protein or FBP) and a noncovalently
bound fluorogen. In the ideal case: (1) neither the FBP nor the fluorogen
exhibits fluorescence in the absence of the other, (2) the increase in
fluorescence upon binding is dramatic, (3) the fluorogenic interaction
between FBP and fluorogen is highly specific, eliminating the need for
washes or blocking agents, (4) neither the FBP nor the fluorogen is toxic
or have intrinsic biological activity, 5) variation of the FBP and/or the
fluorogen elicits useful variation in fluorescence color, binding
affinity and other properties, and 6) the FBP can fold or readjust its
conformation rapidly with an associated fluorescence change.

[0292] For example, such a system may allow the experimenter to modulate
fluorescence as needed by adding or removing fluorogens. Or, fluorogens
could be tailored to address specific requirements, such as cell membrane
permeability and exclusion, or binding to a given FBP with different
affinities and colors to facilitate pulse-chase techniques. Unrelated
fluorogen-FBP pairs that do not cross-react could be developed to support
FRET applications.

[0293] As proof-of-principle, it was demonstrated that cells expressing
single chain antibodies (scFvs) display intense fluorescence enhancement
after exposure to two unrelated dyes, TO1 and MG. ScFvs were chosen
because these small (<30 kDa) molecules retain the full range of
antigen recognition capabilities of the humoral antibodies and are
amenable to use as recombinant tags in fusion proteins. A complex human
scFv library composed of ˜109 synthetically recombined heavy
and light chain variable regions was available in a yeast surface-display
format, enabling us to use Fluorescence Activated Cell Sorting (FACS) to
directly screen for fluorogenic binding to the dyes.

[0294] TO1 and MG are known fluorogens; strong fluorescence activation is
observed when TO1 intercalates into DNA or when MG binds to a specific
RNA aptamer. Without being bound by theory, enhanced fluorescence is
thought to occur because rapid rotation around a single bond within the
chromophore is constrained upon binding. Enhanced fluorescence of such
`molecular rotors` has also been reported for an antibody-dye complex,
although with comparatively modest increase.

[0295] TO1 and MG were coupled to 3350 or 5000 MW polyethylene glycol
(PEG)-biotin, and the dye-PEG-biotin conjugates were used with
streptavidin and anti-biotin magnetic beads to affinity enrich the yeast
surface display library for dye-binding scFvs. The TO1- and MG-affinity
enriched scFv libraries were further enriched and then screened for
fluorescence-generating scFvs by 2-4 rounds of FACS using the dye-PEG
conjugates. For subsequent binding studies, TO1 and MG were coupled to
diethylene glycol diamine (TO1-2p and MG-2p) to maintain antigenic
structure and aqueous dye solubility.

[0296] Sixteen clones that enhanced MG-2p fluorescence and two clones that
enhanced TO1-2p fluorescence were isolated from the library (FIG. 8).
Sequence analysis revealed that the TO1-2p scFvs were encoded by
different heavy and light chain germline genes. The fluorogenic MG-2p
scFvs represented six germline configurations, three composed of the
usual heavy and light chain segments, and three composed of only a single
heavy or light chain segment (FIG. 9). The 11.5-14.4 kD single chain
species are about half the size of GFP (26.7 kD). When expressed on the
yeast cell surface, spectra of the fluorescent scFvs could be determined
in a 96 well homogenous assay format in the presence of free dye.

[0297] We took advantage of the robust surface expression to spectrally
characterize and determine the affinity of all of our FBPs when bound to
these two fluorogens (FIG. 9). The dissociation constants for HLI-TO1 and
HL2-TO1 were high nanomolar range. To obtain stronger binders, one of the
clones (HL1-TO1) was affinity matured by directed evolution using two
rounds of error prone PCR mutagenesis and FACS selection for increased
fluorescence at low fluorogen concentration, generating several FBPs with
improved properties (FIG. 8). The most improved FBP, HL 1.0.1-TO1, bound
TO 1-2p with a cell surface KD of about 3 nM. HL 1.0.1-TO1 and
HL2-TO1 each exhibited modest red excitation shifts (509 and 515 nm,
respectively) relative to free dye absorbance (504 nm) but differed
significantly from one another in emission maxima (530 and 550 nm.)

[0299] To more rigorously investigate the properties of FBP/fluorogens,
secreted forms of HL1.0.1-TO1, HL4-MG and L5-MG were produced and
affinity-purified (FIG. 8). In solution HL1.0.1-TO1 bound TO1-2p with a
KD very similar to that observed on the cell surface. Direct
measurement showed that the fluorescence of TO1-2p increased about
2,600-fold upon binding to the HL 1.0.1-TO1. The extinction coefficient
and quantum yield of the HL1.0.1-TO1/TO1-2p complex (Σ=60,000
M-1

cm-1 and O=0.47) are comparable to the values for Aequorea EGFP
(53,000 and 0.60), and predict that this FBP/fluorogen has EGFP-like
brightness. HL4-MG and L5-MG respectively showed 185- and 265-fold
reduced affinity for MG-2p in solution as compared to surface display but
affinity of H6-ME was reduced only five-fold (FIG. 9). This behavior
differs markedly from that of HL1.0.1-TOL. The quantum yield of the
HL4-MG/MG-2p complex (0.17) is similar to the quantum yield (0.187) of
the malachite green RNA aptamer. Our result reflects a fluorescence
enhancement of about 18,000-fold as compared to free fluorogen, which is
much greater than the 40 to 100-fold enhancement for other
antibody/fluorogen complexes but less than the 50,000-fold increase
observed when a quenched F1AsH-EDT2 reagent binds its cognate
tetracysteine peptide. Absorbance of the FBP/fluorogen is more than
1.4-fold greater than free MG-2p, corresponding to an extinction
coefficient of about 105,000 M-1 cm-1. The combined absorbance
and quantum yield predict a red fluorescent probe with high brightness.

[0300] We have synthesized and obtained several derivatives of the MG
fluorogen to explore whether fluorogenic properties can be usefully
modulated by altered chemistry, and have observed among our six MG FBPS
many changes in binding affinity, fluorescence intensity and
excitation/emission spectra. FIG. 10B illustrates striking spectral
changes produced by 3 MG fluorogen derivatives. It can be seen that a
given fluorogen derivative tends to shift the spectrum to the blue or the
red within a spectral context established by the FBP. However, in some
cases for some fluorogens, specific features of the FBP can greatly alter
the extent of this shift or generate new spectral bands. Malachite green
and most derivatives have a secondary absorbance peak at near ultraviolet
to blue wavelengths that is sensitive to fluorogenic modulation by the
FBP. We have taken advantage of this behavior to demonstrate fluorescent
reporting in two colors excited by a single laser (non-overlapping green
and red colors singly excited by the 488 nm argon laser.) It was found
that color is a property of the combined FBP/fluorogen. Protein context
thus changes the fluorescent behavior of these fluorogens, as has also
been shown for stilbenes.

[0301] It has been long established that certain dyes exhibit enhanced
fluorescence upon binding to proteins, and there are other reports of
fluorogenic binding of dye to an antibody. Several features of what we
report are new and provide promise for development of a new generation of
biosensor systems and live-cell assays. Unlike fluorogen-activating
monoclonal antibodies previously described, the fluorogen-activating
scFvs described here are relatively small and compact monomeric proteins
that can be recombinantly manipulated and expressed, making scFvs suited
for use as genetically expressed tags or as injectable sensors. Unlike
GFP and related fluorescent proteins, the reversibly bound fluorogen
chromophore is directly accessible to experiment and its chemistry can be
modified to alter reporting and sensing capabilities. In particular,
control of access to the dye can afford a new level of selectivity.
Unlike the biarsenical target peptide and the enzymatic peptide tags,
scFvs provide a rich well-understood source of binding variation, which
can be exploited to clone new FBPs that bind new fluorogens, or to
enhance the functionality of existing FBPs and fluorogen variants using
directed evolution technology.

[0302] These studies demonstrated that FBP/fluorogen reporters are well
suited for expression at the extracellular side of the cell membrane.
Although scFvs are derived from extracellular antibodies and contain
internal disulfide linkages, it has been shown that functional scFvs can
be expressed intracellularly in a disulfide-free format. In addition, the
FBP concept can be extended to protein scaffolds beyond scFvs, as has
been done for dye binding motifs such as the rhodamine binding fluorettes
and other structures.

Example 5

Materials and Methods for Example 5

Yeast Display Library

[0303] A yeast cell surface display library, composed of ˜109
recombinant human scFv's derived from cDNA representing a naive germline
repertoire, is obtained from Pacific Northwest National Laboratory
(PNNL). The original version of the library was obtained from PNNL and
was the source of our first isolated FBP, HL 1 TO 1. However, this
library was subsequently found to be contaminated by a low level of
another yeast strain (Candida parapsilosis) that overwhelmed yeast
cultures after repeated outgrowth steps. We obtained another library that
represents a subset of the original PNNL library. The estimated
complexity of this library is ˜8×108 independent scFvs.
This library shows no evidence of contamination, and was the source of
all other FBPS isolated for this study. This uncontaminated library
version is currently available from PNNL.

Yeast Strains

[0304] EBY100 was host to the yeast display library and YVH10 was used to
secrete scFvs, as described. For studies of individual FBPs, pPNL6
plasmids were transferred to JAR200, a G418 resistant derivative of
EBY100. JAR200 expressed higher levels of displayed scFvs and gave higher
transformation rates with pPNL6 plasmids than EBY100.

[0306] All FBPs other than HL1-TOI (see below) were cloned essentially as
described except that a 2-color FACS enrichment screen based on enhanced
fluorescence of the fluorogen was employed instead of a 2-color screen
based on antigen labeled with independent fluorophore. FACS enrichment
was carried out on a Becton Dickinson FACSVantage SE with FACSDiva
option; candidate FBPS were autocloned onto agar plates prior to
characterization. 1 μM TO1-PEG5000-biotin or 500 nM MG-PEG5000biotin
were used to magnetically enrich and sort for respective FBPS, except
that 50 nM MG-PEG5000-biotin was used to autoclone highest affinity
MG-FBP candidates.

Cloning of HL1-TOI

[0307] A large population of induced cells was directly enriched for FBPS
by 3 successive rounds of FACS. Briefly, cells were enriched for affinity
by two rounds of magnetic bead treatment, and the output cells grown and
induced. 108 of these cells were sorted on a MoFlo high speed FACS,
and the output 9×106 cells were immediately resorted under the
same conditions to give 7×104 cells. These cells were again
sorted to give 1500 cells as final output. After growth and induction,
these cells were sorted on an Epics Elite FACS, and the small population
of cells (˜0.5%) with significantly improved fluorogen signal was
collected, regrown and resorted. These cells exclusively displayed
HL1-TO1. Subsequent attempts at cloning other FBPs using this direct
approach failed.

Identification of FBPs

[0308] Autocloned yeast cells displaying candidate FBP isolates were grown
in small cultures, and yeast plasmid DNA isolated using a Zymoprep kit
(Zymo Research). The scFv insert was PCR amplified, and the amplified DNA
product purified on an agarose gel and then DNA sequenced. scFv variable
region sequences were classified as to human germline composition by
analysis on the IMGTN-QUEST website.

Spectral Characterization of FBPs

[0309] Yeast surface displayed scFvs were spectrally characterized using
fluorescence bottom reading in 96 well microplates on a Tecan Safire2
plate reader. 106 cells in 200 μl yeast buffer were assayed with
100-1000 nM MG-2p or TO1-2p. Spectra were corrected by subtraction of
fluorescence of control cells not expressing scFvs.

Affinity Maturation of HL1-TO1

[0310] Affinity maturation of HL1-TO1 followed described methods for
random mutagenesis and selection of improved clones, except that the
2-color FACS screen used in our standard cloning procedure was employed
using TO1-2p as the fluorogen.

Secretion and Purification of Soluble FBPs

[0311] Induction and secretion of scFvs were at 20° or 25°
C. as described, except that YEPD was replaced by a tryptone-based
secretion medium:

[0313] Culture supernatants were dialysed and concentrated 3 times against
6 liters PBS on an Amicon Model 2000 high performance ultrafiltration
cell using a 10,000 mw cut-off cellulose membrane. To purify the 6-his
tagged FBPS, the concentrated dialysate (˜50 ml) was subjected to
nickel-nitrilotriacetic acid chromatography (Ni-NTA) according to
manufacturer's instructions. Appropriate dilutions of eluted fractions
were assayed for fluorogenic activity using essentially the same assay as
for secretion. Fluorescent fractions were pooled, assayed for protein
content using a BCA protein assay kit, and analyzed by SDS gel
electrophoresis.

[0314] A homogenous assay under equilibrium binding conditions was devised
to determine the binding affinity of fluorogen to yeast displayed scFvs.
A flow cytometric method for titrating yeast displayed scFvs with
fluorescently labeled antigen was adapted to the use of fluorogens.
106 induced yeast in 200 μl modified PBS (˜1 nM displayed
scFvs) containing fluorogen over a concentration range of 0.1-1000 nM
were assayed in duplicate for fluorescence in 96 well microplates on a
Tecan Safire2 reader. As controls, mock induced JAR200 cells that do not
express scFvs were treated with equal concentrations of fluorogen;
fluorescence was corrected by subtraction of the fluorescence of control
cells. Cell surface KD values were determined on Prism Graphpad
Prism 4.0 software by non-linear regression analysis using a one-site
binding algorithm for saturation binding:

Y═Bmax*X═(KD+X)

where X is the concentration of fluorogen.

Determination of Fluorogen Binding Affinity to Soluble FBPs

[0315] Binding affinity to soluble scFvs was determined by monitoring
fluorogenic signal under conditions of ligand depletion using a
homogenous 96 well microplate assay similar to above. 1 nM HL1.0.1-TO1,
10 nM L5-MG and 100 nM HL4-MG were each assayed with a 0.1 to 1000 nM
range of fluorogen. Fluorescence of each FBP+dye sample was corrected by
subtracting the fluorescence of a dye only sample. KD values were
determined by non-linear regression using Graphpad Prism 4.0 and a ligand
depletion algorithm.

Y═(X+KD+R- {square root over ((-X--KD-R)2-4*X*R)})/2

where X is the concentration of fluorogen, and R is the concentration of
FBP/fluorogencomplex at the observed or extrapolated plateau at maximum
fluorescence.

Determination of Quantum Yields

[0316] Quantum yields were determined by comparing integrated spectra of
FBP/fluorogen complexes with those of reference dyes. Corrected emission
spectra were taken at concentrations of FBP/fluorogen complex and
reference dyes giving similar absorbances at the excitation wavelength,
and the intensity integral computed. Provisional quantum yields were
calculated by the relation:

Φ = Φ R * I * A R * η 2 I R * A * η R 2
##EQU00001##

where (phi is the quantum yield, I is the integrated intensity, A is the
absorbance, η is the refractive index, and R designates the reference
dye. Provisional quantum yields were adjusted to 1:1 complexation by
using the solution KD of the complex to quantify the proportional
occupancy at these concentrations (using Graphpad Prism 4.0 and the above
ligand depletion algorithm).

[0317] A cyanic dye, Cy5.18 in PBS and Di-S--C2-(5) in MeOH were used as
reference fluorophores for the determination of HL4-MG/MG-2p and
L5-MG/MG-2p quantum yields. 2 μM scFv and 440 nM MG-2p were employed;
emission spectra of respective complexes were taken in duplicate.
Absorbances of the respective complexes were significantly higher than
free MG-2p. These differences in absorbance magnitude underrepresent
actual differences because of incomplete complexation at these
concentrations. HL4MG/MG-2p and L5-MG/MG-2p quantum yields were
respectively multiplied by 1.35 and 1.20 to correct for incomplete
complexation. Fluorescein in 0.1 N NaOH and Rhodamine-6-G in MeOH were
used as reference fluorophores for the determination of the quantum yield
of HL1.0.1-TO1/TO12p. At the employed concentrations of 2 μM scFv and
520 nM TO1-2p, virtually complete complexation is expected, and no
correction was used. Only slight changes in absorbance magnitude were
noted.

Determination of Fluorogenic Enhancement

[0318] Fluorogenic enhancement of HL4-MG, L5-MG and HL1.0.1-TO1 was
measured in 96 well microplates on a Tecan Safire2 reader by comparison
of the fluorescence of 500 nM free fluorogen with the fluorescence of a
mixture of 500 nM fluorogen and 2 μM respective FBP. After a 1 hour
incubation to allow complex formation, fluorescence was measured at the
excitation and emission maxima of the fluorogen/FBP complexes.
Fluorescence of yeast PBS buffer was subtracted from all samples.
Fluorescence readings were stable for at least 16 hours.

[0319] Extended gain settings were used to increase numerical accuracy.
For HL4-MG and L5MG, the differences in fluorescence between complexes
and free dye exceeded the extended gain range of the instrument, so
intermediate concentrations of Cy5 were also measured to allow
interpolation between the extreme values. Two independent experiments,
each with triplicate measurements, were carried out for each of the 3
FBPS; standard deviations for averaged values of FBPS, free fluorogen or
PBS were less than 5%. The fold-enhancement values for HL4-MG and L5-MG
were respectively multiplied by 1.35 and 1.20 to correct for partial
complexation.

Mammalian Cell-Surface Expression of FBP Molecules

[0320] Plasmids expressing surface-displayed scFv's were generated as
follows. A 375 by PCRamplicon was amplified from E. coli C600 DNA using
as primers:

This molecule, which contains the lac promoter and 271 nucleotides of
beta-galactosidase coding sequence flanked by SfiI sites, was cut with
XmaI and ligated into pDisplay (Invitrogen) between the Sma1 and XmaI
sites to produce vector pDisplayBlue. Individual scFv sequences were
prepared for insertion between the SfiI sites in pDisplayBlue by
PCR-amplifying the scFv sequences from pPNL6 clones using as primers:

These amplicons were cut with SfiI, ligated into SfiI-cut vector,
transformed into DHScc E. coli to ampicillin resistance, and Lac+
colonies picked for DNA sequencing. DNA was prepared from selected
transformants using Qiagen Mini-Prep kits and transfected into NIH3T3
cells or M21 mouse melanoma cells (˜1 μg DNA per 105 cells)
in 24-wellplates using Lipofectamine 2000 (Invitrogen) following the
protocols supplied by the manufacturer. Stable transfectants were
isolated by successive rounds of FACS sorting of cells exposed to the
appropriate fluorogens.

Example 6

Fusion of scFv Sequences to a Transmembrane Domain Derived from Human
Fibroblast Growth Factor Receptor 2, and Expression of the Fusion
Constructs in Mammalian Cells

[0321] NIH3T3 cells stably expressing HL4-MG fused to PDGFR were imaged by
confocal microscope at 633 nm excitation after treatment for 5 min in PBS
with 200 nM MG-11p or 200 nM MG-ester. On longer incubation, MG-ester
illuminated intracellular features such as the nuclear periphery
(endoplasmic reticulum) and Golgi become more difficult to visualize.
Fluorescence images were excited at 633 nm using a 650 nm long pass
filter. Fluorescence images were unprocessed; interference lines on DIC
images were removed using a fourier transform filter in Photoshop. Images
in were acquired on a Zeiss LSM510 META laser scanning microscope using a
63× objective. Fluorescence images were excited at 633 nm using a
650 nm long pass filter.

[0324] In another experiment, it was shown that simultaneous surface
labeling of fibroblasts with MG and TO1 FAPs occurred. NIH3T3 cells
respectively expressing the FAPs 1:1 and imaged using 10 nM MG-2p and 40
nM TO1-2p. The transparency of surface-labeled cells allows fine
discrimination of contact surfaces between cells of different colors.
Images were acquired on a Zeiss Axioplan 2 with Apotome microscope. Green
false color (TO1-2p) was imaged using 540/25 and 605/55 nm excitation and
emission filters; red false color was imaged using 560/55 and 710/75 nm
excitation and emission filters. Images were reconstructed from 72 1
μm sections, displayed as 15 projections in NIH Image software, and
false colored in Adobe Photoshop. a 700/75 nm bandpass filter was used to
visualize the 488 nm excitation of HL4-MG

Example 7

Fusion of scFv Sequences to the Human Glucose Transporter GLUT4, and
Expression of the Fusion Constructs in Mammalian Cells

[0325] An open reading frame comprising the coding sequence of human GLUT4
was cloned 5' to the PDGFR sequence in the modified pDisplay vector
described in Example 5 that was further modified to accept the insert
between PflMI restriction sites. Fluorogen-activating scFv sequence
HL1.1-TO1 was cloned between the SfiI sites of these constructs as
described in Example 5. NIH3T3-L1 cells were transfected with the
constructs, and stable transfectants were isolated by multiple rounds of
FACS sorting for fluorogen-activating cells.

[0326] Imaging by fluorescence microscopy after incubation in the presence
of fluorogen showed distinct surface labeling when cells were treated
with a membrane impermeant fluorogen, and internal as well as surface
labeling when cells were transfected with a control plasmid with EGFP
cloned between the SfiI sites.

[0327] Images were taken with Apotome microscope with 63× water
immersion lens. Excitation occurred at 488 nm with emission collected in
GFP channel. Cells are undifferentiated 3T3-L1 fibroblasts. These results
indicate that a fusion protein between GLUT4 and a fluorogen-activating
scFv is correctly localized at the cell surface, as well as in the
expected subcellular compartments of the endomembrane system.

Example 8

Fusion of scFv Sequences to the Human G-Protein Coupled Receptor ADRB2,
and Expression of the Fusion Constructs in Mammalian Cells

[0328] The coding sequence of human ADRB2 was PCR amplified from an ADRB2
fosmid and cloned into the BsmI site in the modified pDisplay vector
described in example N, with an in frame stop codon at the 3' end of the
ADRB2 sequence. Fluorogen-activating scFv sequences HL1.1-TO1 and HL4MG
were cloned into the SfiI sites of these constructs as described in
example N. NIH3T3 cells were transfected with the constructs, and stable
transfectants were isolated by multiple rounds of FACS sorting for
fluorogen-activating cells.

[0329] Imaging by fluorescence microscopy after incubation in the presence
of fluorogen showed distinct surface labeling when cells were treated
with membrane impermeant fluorogens, and internal as well as surface
labeling when cells were treated with membrane permeant fluorogens.
Further, when cells were treated with the ADRB2 agonist isoproterenol at
physiological concentrations, internalization of the ADRB fusion protein
was observed, indicating that the fusion protein was physiologically
active with respect to recognition and internalization of the agonist.

Example 9

Cell Surface Complementation

[0330] This experiment demonstrated that the light chain and heavy chains
of a selected scFv can be separately fused to two different proteins, and
that when these two proteins are in close proximity, the heavy and light
chains associate to produce a binding site for the same dye that binds to
the original scFv leading to a fluorescence increase. In the following
experiments three classes of yeast cells were generated by molecular
biology methods known in the art. One class of yeast expressed only the
heavy chain of the scFv on the surface. A second class expressed only the
light chain of the scFv on its surface. The third class expressed both
the heavy and light chains on the surface. When the fluorescent reporter
TO1 was added independently to solutions containing the three classes of
yeast cells, only the third class that expressed both heavy and light
chains at a high surface density produced a fluorescence increase, as
shown below. Specifically, two vectors, pPNL6 and pPNL6URA3 were
prepared, where the TRP1 gene of pPNL6 was replaced with the URA3 gene of
S. cerevisiae. This approach allowed selection for both plasmids in a
single cell. Fragments carrying scFv1, scFv1 HO (heavy only chain), or
scFv1 LO (light only chain) were cloned into each of the two vectors.

[0331] JAR200 yeast cells were transformed with each single plasmid as
well as both scFv1 HO (PNL6)+scFv1 LO (PNL6URA3) or both scFv1 HO
(PNL6URA3)+scFv1 LO (PNL6). Analysis by FACS and TECAN followed induction
of the cells. In both cases, 1 μM TO1-2P was used.

[0332] In the first approach, flow cytometry was used to assay cellular
fluorescence of the three classes of yeast in the presence and absence of
TO 1. The population of induced cells was analyzed by measuring
fluorescence emitted at 685 from a signal caused by protein labeled by
anti-C-myc Alexa 647. This allowed confirmation that the three classes of
yeast cells were expressing full length heavy, light, or heavy and light
chains on the cell surface. For cells that expressed full length
fragments the TO1-2p fluorescence was observed as emission at 530.

[0333] The cells expressing both heavy and light were 2.5-3 times more
fluorescent than cells expressing heavy or light only. The signal
increase is likely to be larger in assays where the two proteins
containing the heavy and light chains are associating in a pair-wise
manner rather that a statistical association. With pair-wise
protein-protein interactions the fluorescence of the complex may approach
that of the original scFv PNL6 or PNL6URA3 in which the heavy and light
chains are directly linked through a short-serine-glycine-polymeric
linker.

[0334] In the second approach, a fluorescence spectrometer was used to
quantify the fluorescence from suspensions of cells. The fluorescence
excitation wavelength was 506 nm and the emission wavelength was 610 nm.
Values presented below are corrected from raw data by subtracting out the
values for (buffer+dye) and induced (no dye) samples. They were then
corrected for the per cent of the population that was induced, as
determined by the flow cytometry analysis.

[0344] 2-[(1-(3-Aminopropyl)-4(1H)-quinolinylidene)methyl]-3-(3-sulfopropy-
l)benzothiazolinium hydrochloride TO1 4 (5.8 mg; 0.01 mmol) was dissolved
in 0.1 mL of water. Biotin-PEG5000-NHS ester 8 (Nektar Therapeutics) (50
mg; 0.01 mmol) dissolved in 0.1 mL of DMF was added to the TO1 solution
followed by 0.1 mL of saturated sodium bicarbonate solution. The reaction
mixture was stirred for 1 hr. The solvents were removed under vacuum and
the residue was taken up in a minimum of water and passed through a P4
sized exclusion column to remove free TO 1. The PEG fraction was
concentrated and purified on Q-Sepharose (Amersham Biosciences) to
separate unlabeled Biotin-PEG from Biotin-PEG5000-TO1. MS: Mn
5743.56;

[0349] [4-(Boc-9-amino-6-aza-1-oxa-5-oxo-nonyl)phenyl]bis[4-(dimethylamino-
)phenylmethane (118 mg, 0.2 mmol) was dissolved in HCl/ethanol (2 mL of a
5% solution). The reaction mixture was kept overnight at room
temperature. The solvent was removed and the residue dried. The product
was used as such in the next reaction step.

[0354] MG-11p 20 (11 mg; 0.01 mmol) was dissolved in 0.2 mL DMSO. A
solution of BiotinNHS ester (6.5 mg; 0.02 mmol) in 0.1 mL DMSO was added
followed by 0.02 ml, of DEA (1 mMol in DMSO). The reaction mixture was
stirred for 2 hrs at room temperature, then passed through a short column
of neutral aluminum oxide. DMSO and DEA were eluted with chloroform. The
product was eluted with chloroform/10-20% methanol to give 8 mg of a
green solid. Yield: 58%.

[0356] MG-2P 18 (5 mg; 0.01 mmol) and Biotin-PEG5000-NHS ester 8 (Nektar
Therapeutics) (50 mg; 0.01 mmol) were dissolved in 0.2 mL of DMF and 0.01
mL of DEA (1 mMol in DMF) was added. The reaction mixture was stirred for
1 hr. The reaction mixture was passed through a short column of neutral
aluminum oxide. DMF and DEA were eluted with chloroform. The product was
eluted with chloroform/10-20% methanol. Mn=5747.8.

[0358] All publications and patents mentioned herein, included those
listed below, are hereby incorporated by reference in their entirety as
if each individual publication or patent was specifically and
individually incorporated by reference. In case of conflict, the present
application, including any definitions herein, will control.

[0360] Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. While specific
embodiments of the subject invention have been discussed, the above
specification is illustrative and not restrictive. Many variations of the
invention will become apparent to those skilled in the art upon review of
this specification. The full scope of the invention should be determined
by reference to the claims, along with their full scope of equivalents,
and the specification, along with such variations. Such equivalents are
intended to be encompassed by the following claims.